Compositions and methods for repetitive use of genomic DNA

ABSTRACT

The present invention relates to detection or genotyping (or other sample analysis) of target nucleic acids following immobilization of the target nucleic acids onto a surface. The target nucleic acids can be re-used multiple times, thus conserving sample materials and simplifying sample preparation.

FIELD OF THE INVENTION

[0001] The present invention relates to detection or genotyping (orother sample analysis) of target nucleic acids following immobilizationof the target nucleic acids onto a surface. The target nucleic acids canbe re-used multiple times, thus conserving sample materials andsimplifying sample preparation.

BACKGROUND OF THE INVENTION

[0002] The detection of specific nucleic acids is an important tool fordiagnostic medicine, molecular biology research and forensic analysis.Gene probe assays currently play roles in identifying infectiousorganisms such as bacteria and viruses, in probing the expression ofnormal and mutant genes and identifying mutant genes such as oncogenes,in typing tissue for compatibility preceding tissue transplantation, inmatching tissue or blood samples for forensic medicine, and forexploring homology among genes from different species.

[0003] Ideally, a gene probe assay should be sensitive, specific andeasily automatable (for a review, see Nickerson, Current Opinion inBiotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to amplify exponentially a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, 4:41-47 (1993)).

[0004] Specificity, in contrast, remains a problem in many currentlyavailable gene probe assays. The extent of molecular complementaritybetween probe and target defines the specificity of the interaction.Variations in the concentrations of probes, of targets and of salts inthe hybridization medium, in the reaction temperature, and in the lengthof the probe may alter or influence the specificity of the probe/targetinteraction.

[0005] It may be possible under some circumstances to distinguishtargets with perfect complementarity from targets with mismatches,although this is generally very difficult using traditional technology,since small variations in the reaction conditions will alter thehybridization. New experimental techniques for mismatch detection withstandard probes include DNA ligation assays where single pointmismatches prevent ligation.

[0006] Recent focus has been on the analysis of the relationship betweengenetic variation and phenotype by making use of polymorphic DNAmarkers. Previous work utilized short tandem repeats (STRs) aspolymorphic positional markers; however, recent focus is on the use ofsingle nucleotide polymorphisms (SNPs), which occur at an averagefrequency of more than 1 per kilobase in human genomic DNA. Some SNPs,particularly those in and around coding sequences, are likely to be thedirect cause of therapeutically relevant phenotypic variants and/ordisease predisposition. There are a number of well known polymorphismsthat cause clinically important phenotypes; for example the apoE2/3/4variants are associated with different relative risk of Alzheimer's andother diseases (see Cordor et al., Science 261(1993). Multiplex PCRamplification of SNP loci with subsequent hybridization tooligonucleotide arrays has been shown to be an accurate and reliablemethod of simultaneously genotyping at least hundreds of SNPs; see Wanget al., Science, 280:1077 (1998); see also Schafer et al., NatureBiotechnology 16:33-39 (1998).

[0007] However, difficulty has been encountered in obtaining significantdata in large-scale genotyping or SNP identification studies because thetarget or sample is consumed during the assay. Accordingly, there existsa need for a method of reusing target nucleic acids, in particular,target genomic DNA.

[0008] There are a variety of particular techniques that are used todetect sequence, including mutations and SNPs. These include, but arenot limited to, ligation based assays, single base extension methods(see WO 92/15712, EP 0 371 437 B1, EP 0317 074 B1; Pastinen et al.,Genome Res. 7:606-614 (1997); Syvanen, Clinica Chimica Acta 226:225-236(1994); and WO 91/13075), cleavage based assays such as Invader™technology, Q-Beta replicase (QβR) technology and competitive probeanalysis (e.g. competitive sequencing by hybridization; see below).

[0009] Oligonucleotide ligation amplification (“OLA”, which is referredas the ligation chain reaction (LCR) when two-stranded reactions)involves the ligation of two smaller probes into a single long probe,using the target sequence as the template. See generally U.S. Pat. Nos.5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP0 439 182 B1; WO 90/01069; WO 89/12696; WO 97/31256 and WO 89/09835, allof which are incorporated by reference.

[0010] An additional technique utilizes sequencing by hybridization. Forexample, sequencing by hybridization has been described (Drmanac et al.,Genomics 4:114 (1989); Koster et al., Nature Biotechnology 14:1123(1996); U.S. Pat. Nos. 5,525,464; 5,202,231 and 5,695,940, among others,all of which are hereby expressly incorporated by reference in theirentirety).

[0011] PCTs US98/21193, PCT US99/14387 and PCT US98/05025; WO98/50782;and U.S. Ser. Nos. 09/287,573, 09/151,877, 09/256,943, 09/316,154, No.60/119,323, Ser. No. 09/315,584; all of which are expressly incorporatedby reference, describe novel compositions utilizing substrates withmicrosphere arrays, which allow for novel detection methods of nucleicacid hybridization.

[0012] Samples can be scarce and difficult to obtain. Reusing target DNAcan be critical in large studies such as SNP genotyping studies, toreduce DNA consumption and sample preparation costs. None of the currentmethods allow the rapid, facile, repeated and inexpensive analysis of atarget nucleic acid by re-using the target after it is immobilized on asubstrate. Accordingly, it is an object of the present invention toprovide methods and compositions for such determinations.

SUMMARY OF THE INVENTION

[0013] In accordance with the objects outlined above, the presentinvention provides composition comprising a substrate comprisingimmobilized target nucleic acids. The target nucleic acids comprisegenomic DNA and an attachment moiety for attaching the genomic DNA tothe substrate, wherein the attachment moiety is capable of withstandingmultiple analyses of the genomic DNA.

[0014] In addition the invention provides a method comprising providinga substrate comprising a plurality of immobilized genomic DNA targetsequences, performing a first analysis of the target sequences wherebythe immobilized genomic DNA is not consumed and performing a secondanalysis of the immobilized genomic DNA target sequences. In a preferredembodiment the first and second analyses are genotyping analyses and canalso include amplification reactions.

[0015] In addition the invention provides a method comprising providinga substrate comprising a plurality of immobilized genomic DNA sequences,hybridizing the genomic DNA sequences with a first set of ligationprimers to form first ligation complexes, whereby the first ligationprimers hybridize to the genomic DNA sequences flanking a first targetsequence. The method further includes removing unhybridized ligationprimers. In addition the method includes contacting the first ligationcomplexes with a ligation enzyme, whereby when the first ligationprimers are complementary to the first target sequences, the ligationenzyme ligates the first ligation primers generating first ligationproducts. In addition the method includes removing the first ligationproducts from said immobilized genomic DNA. The invention furtherincludes hybridizing the genomic DNA with second ligation primers toform second ligation complexes, whereby the second ligation primershybridize to the genomic DNA sequences flanking a second targetsequence, contacting the second ligation complex with a ligation enzyme,whereby when the second ligation primers are complementary to the secondtarget sequence, the ligation enzyme ligates said second ligationprimers generating second ligation products. The method also includesdetection the ligation products.

[0016] In addition the invention provides a method comprising providinga composition comprising first primers and target nucleic acid andperforming a first analysis of the target nucleic acid wherein the firstanalysis includes contacting the first primers with the target nucleicacid whereby at least one of the first primers hybridizes with thetarget nucleic acid. Further the method includes removing unhybridizedfirst primers and contacting the hybridized first primers with an enzymesuch that the hybridized first primers are modified forming firstmodified primers, whereby the target nucleic acid is not consumed, andperforming a second analysis of the target nucleic acid.

[0017] In addition the invention includes amplifying the products of thegenotyping reaction, i.e. the ligation reaction and detecting theamplicons. Detection is accomplished by a variety of methods includingmass spectrometry, flow cytometry, ordered arrays, random arrays,capillary electrophoresis and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 depicts competitive hybridization of target probes (31, 32,33, 41, 42, 43, 51, 52 and 53) to target sequences (21, 22, and 23)immobilized on a surface 10. The target probes comprise labels (star,triangle and square). Following hybridization, unbound probes areremoved from the sample; bound probes are subsequently released anddetected. Thus, the samples of target material can be reused.

[0019]FIG. 2 depicts single base extension (SBE) assay to detect thebase at a detection position 25 in a target nucleic acid 20 that isimmobilized to a solid support 10. A set of target probes 32, 33 arecontacted with the immobilized target. The unbound probes are removed.Labeled nucleotides and polymerase are added to the complex. A labelednucleotide 35 that is complementary to the nucleotide at the detectionposition is incorporated into the target probe 32. The hybridizedlabeled probe is removed. Now the sample of target nucleic acids can bereused.

[0020]FIG. 3 depicts SBE followed by oligonucleotide ligation assay(OLA). Two probes, an upstream 44 and downstream 42 probe are hybridizedwith target nucleic acid immobilized on a surface 10. The target nucleicacid includes an upstream target sequence 24, a detection position 25and a downstream target sequence 26. Following hybridization of theprobes 42 and 44 to the target nucleic acid, a polymerase andnucleotides that can be optionally labeled are contacted with thehybridization complex. A nucleotide that is complementary to thenucleotide at the detection position is incorporated into the upstreamprobe 44. Non-extended probes are removed. A ligase is contacted withthe hybridization complex and ligates the modified (extended) upstreamprobe 44 with the downstream probe 42 to form a modified target probe47. The modified target probe is removed from the immobilized targetnucleic acid and the immobilized target nucleic acid is re-used.

[0021]FIG. 4 depicts the oligonucleotide ligation assay (OLA). Upstreamand downstream ligation probes 42 and 44 are hybridized to targetnucleic acids immobilized to a solid support. The target nucleic acidcomprises upstream and downstream target sequences 24 and 26,respectively, and a detection position 25. The upstream ligation probescomprise a nucleotide that is complementary to the nucleotide at thedetection position 45. The set of upstream ligation probes includesnucleotides that are complementary 45 and those that are not 46.Following hybridization of the probes to the target nucleic acid, theprobes that are not complementary at the detection position are removed.Ligase is added. The ligated probes are removed from the immobilizedtarget nucleic acid and detected by various methods including flowcytometry, ordered or positioned arrays, capillary electrophoresis ormass spectrometry. The immobilized target nucleic acid is then re-used.

[0022]FIG. 5 demonstrates that solid phase genomic DNA is reusable.

[0023]FIG. 6 demonstrates that genomic DNA on magnetic particles isreusable in the oligonucleotide ligation assay. A. OLA assay with 48Loci. There are two negative controls, (A and B) and two positivecontrols, (C and D). B. OLA assay with 48 Loci. The same ‘beaded’ DNA isused the next day. There are two negative controls, (A and C) and twopositive controls, (B and D).

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is directed to methods of amplification andgenotyping, and in particular to methods that include the re-use ofgenomic DNA in genotyping and amplification assays. The invention can beused with adapter sequences that include universal primers. Products ofgenotyping reactions are detected by a variety of methods including flowcytometry, positioned arrays, capillary electrophoresis, massspectrometry or bead arrays as described herein.

[0025] The invention can generally be described as follows. A pluralityof probes (sometimes referred to herein as “target probes”) arecontacted with sample genomic DNA that is at some point immobilized to asolid support. That is, the sample can be immobilized either prior to orafter hybridization with the probes. Preferably the target probes aredesigned to have a first portion that is target-specific and two“priming” portions, an upstream and downstream priming sequence, thatflank the target specific portion. These priming sequences arepreferably “universal”; that is, all target probes have the same primingsequences. These target probes are hybridized to target sequences toform hybridization complexes. Non-hybridized sequences are then removed.This is generally performed by removing the liquid phase containing thenon-hybridized probes and optionally washing the immobilizedhybridization complexes. Following this step, the sample genomic DNA canbe reused. That is, the genomic DNA is not consumed in the analysis orgenotyping reaction. By “consumed” is meant degraded or otherwisemodified so as to prevent subsequent analysis.

[0026] For direct detection of genomic sequences, the hybridizationcomplexes are denatured, and the target probes collected. Then PCRprimers (although as described herein, other amplification reactions canbe performed) that correspond to the priming sequences are added, andamplification proceeds. Other amplification schemes include but are notlimited to T7 amplification and Invader™ technology. The resultingamplicons, which can be directly or indirectly labeled, can then bedetected. Detection is accomplished by a variety of methods includingflow cytometry, positioned arrays, capillary electrophoresis, massspectrometry or bead arrays as described herein on arrays. This allowsthe detection and quantification of the target sequences.

[0027] In a preferred embodiment, rather than simple detection,genotyping reactions are performed on the immobilized genomic DNA. Avariety of known genotyping reactions can be performed, including, butnot limited to oligo ligation assay (OLA), single-base extension (SBE),allelic PCR (aPCR), and cleavage reactions such as Invader™, and thelike. In each case, the target probe(s) used for the genotyping reactioncontain priming sequences. Preferably, the unhybridized probes areremoved prior to the genotyping reaction. Once the genotyping reactionis complete and unhybridized probes have been removed, the hybridizationcomplexes are denatured, and the target probes are amplified. Theresulting amplicons are then detected as outlined herein.

[0028] One particular strength of the present invention is that theimmobilized genomic DNA can be reused indefinitely. The method finds anotable advantage in the ability to generate multiple genotyping oramplification assay products from a single sample of genomic DNA. Thatis, by reusing genomic DNA, multiple genotypes or amplificationreactions can be performed on a single sample. Thus, for example, afirst set of target probes directed to a first set of SNPs can be added,reacted and removed, and then a second set of target probes added to asecond set of SNPs. This process can be repeated, thus allowing diverseand robust genomic information to be obtained.

[0029] As will be appreciated by those in the art, the system can takeon a wide variety of conformations, depending on the assay. For example,the oligonucleotide ligation assay (OLA) can be performed. OLA relies onthe fact that two adjacently hybridized probes will be ligated togetherby a ligase only if there is perfect complementarity at each of thetermini, i.e. at a detection position. In this embodiment, there are twoligation probes: a first or upstream ligation probe that comprises theupstream priming sequence and a second portion that will hybridize to afirst domain of the target sequence, and a second or downstream ligationprobe that comprises a portion that will hybridize to a second domain ofthe target sequence, adjacent to the first domain, and a second portioncomprising the downstream priming sequence. If perfect complementarityat the junction exists, the ligation occurs and then the resultinghybridization complex (comprising the target and the ligated probe) canbe separated as above from unreacted probes. Again, the priming sitesare used to amplify the ligated probe to form a plurality of ampliconsthat are then detected in a variety of ways, as outlined herein.Alternatively, a variation on this theme utilizes rolling circleamplification (RCA), which requires a single probe whose ends areligated, followed by amplification.

[0030] In addition, any of the above embodiments can utilize one or more“adapter sequences” (sometimes referred to in the art as “zip codes”) toallow the use of “universal arrays”. That is, arrays are used thatcontain capture probes that are not target specific, but rather specificto individual artificial adapter sequences. The adapter sequences areadded to the target probes (in the case of ligation probes, either probemay contain the adapter sequence), nested between the priming sequences,and thus are included in the amplicons. The adapters are then hybridizedto the capture probes on the array, and detection proceeds.

[0031] Accordingly, the present invention may include any appropriatemethods of amplification and detection of target sequences in a sample.Preferably the target sequence is genomic DNA. In addition the method isdirected to re-using target sequences in sequential amplificationreactions or other genotyping analyses or assays.

[0032]

[0033] In a preferred embodiment, the present invention provides methodsand compositions for the repeated use and detection of target sequencesin samples. As will be appreciated by those in the art, the samplesolution may comprise any number of things, including, but not limitedto, forensic samples, bodily fluids (including, but not limited to,blood, urine, serum, lymph, saliva, anal and vaginal secretions,perspiration and semen, of virtually any organism, with mammaliansamples being preferred and human samples being particularly preferred);environmental samples (including, but not limited to, air, agricultural,water and soil samples); biological warfare agent samples; researchsamples (i.e. in the case of nucleic acids, the sample may be theproducts of an amplification reaction, including both target and signalamplification as is generally described in U.S. S. No. 60/161,148;hereby incorporated by reference) such as PCR amplification reaction);purified samples, such as purified genomic DNA, RNA, proteins, etc.; rawsamples (bacteria, virus, genomic DNA, etc.); as will be appreciated bythose in the art, virtually any experimental manipulation may have beendone on the sample.

[0034] The present invention is directed to the repeated detection oftarget sequences from a sample. By “target sequence” or “target nucleicacid” or grammatical equivalents herein means a nucleic acid sequence ona single strand of nucleic acid. The target sequence may be a portion ofa gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA andrRNA, or others. Preferably the target sequence is genomic DNA.

[0035] As is outlined herein, the target sequence may be a targetsequence from a sample that has been amplified. It may be any length,with the understanding that longer sequences are more specific. As willbe appreciated by those in the art, the complementary target sequencemay take many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, readout probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart. Preferably, however, the target sequence is not amplified prior toattachment to the solid support.

[0036] The target sequence may also be comprised of different targetdomains. The target domains may be adjacent or separated as indicated.Unless specified, the terms “first” and “second” are not meant to conferan orientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain.

[0037] By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below (forexample for target probes, etc), nucleic acid analogs are included thatmay have alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J.Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of labels, alter the hybridization properties of thenucleic acids, or to increase the stability and half-life of suchmolecules in physiological environments.

[0038] As will be appreciated by those in the art, all of these nucleicacid analogs may find use in the present invention. In addition,mixtures of naturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occuring nucleic acids and analogs may be made.

[0039] Particularly preferred are peptide nucleic acids (PNA) whichincludes peptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration.

[0040] The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occurring analog structures. Thusfor example the individual units of a peptide nucleic acid, eachcontaining a base, are referred to herein as a nucleoside.

[0041] While generally described for human patients, the compositionsand methods of the invention find use in detection of target sequencesfrom a variety of sources. That is, the “target source” or source oftarget sample need not be limited to patients or even to humans. Indeedthe method finds use in detection of target sequences from any number ofsources including, plants, animals, and microorganisms such as bacteriaand viruses. In a preferred embodiment the source is a mammal includinghumans, domestic and farm animals, and zoo, sports, or pet animals, suchas dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the mammal is human. In addition, the source is a cell type,including prokaryotic or eukaryotic cells. Suitable prokaryotic cellsinclude, but are not limited to, bacteria such as E. coli, Bacillusspecies, and the extremophile bacteria such as thermophiles, etc.Preferably, the procaryotic target cells are recombination competent.Suitable eukaryotic cells include, but are not limited to, fungi such asyeast and filamentous fungi, including species of Aspergillus,Trichoderma, and Neurospora; plant cells including those of corn,sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa,sunflower, etc.; and animal cells, including fish, birds and mammals.Suitable fish cells include, but are not limited to, those from speciesof salmon, trout, tulapia, tuna, carp, flounder, halibut, swordfish, codand zebrafish. Suitable bird cells include, but are not limited to,those of chickens, ducks, quail, pheasants and turkeys, and other junglefowl or game birds. Suitable mammalian cells include, but are notlimited to, cells from horses, cattle, buffalo, deer, sheep, rabbits,rodents such as mice, rats, hamsters, gerbils, and guinea pigs, minks,goats, pigs, primates, marsupials, marine mammals including dolphins andwhales, as well as cell lines, such as human cell lines of any tissue orstem cell type, and stem cells, including pluripotent andnon-pluripotent, and non-human zygotes.

[0042] In addition suitable cell types include, but are not limited to,tumor cells of all types (particularly melanoma, myeloid leukemia,carcinomas of the lung, breast, ovaries, colon, kidney, prostate,pancreas and testes), cardiomyocytes, endothelial cells, epithelialcells, lymphocytes (T-cell and B cell), mast cells, eosinophils,vascular intimal cells, hepatocytes, leukocytes including mononuclearleukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney,liver and myocyte stem cells (for use in screening for differentiationand de-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

[0043] Generally, when the target nucleic acid is genomic DNA, thegenomic DNA is isolated or purified from the sample. Methods forpurifying genomic DNA are known in the art. For example, the sample maybe treated to lyse the cells, using known lysis buffers, sonication,electroporation, etc., with purification and amplification as outlinedbelow occurring as needed, as will be appreciated by those in the art.Once isolated, the genomic DNA is fragmented or sheared as is known.Alternatively, the genomic DNA is cleaved, for example with nucleases.In addition, genomic DNA can be prepared with commercial kits such agenomic DNA preparation kits from Qiagen or Promega, including Wizard™Genomic DNA purification kit or ReadyAmp™ genomic DNA PurificationSystem. Additional methods for preparing genomic DNA are outlined inU.S. Ser. No. 09/785,514, filed Feb. 16, 2001, which is expresslyincorporated herein be reference.

[0044] In one embodiment the genomic DNA (gDNA) is cleaved or sheared.Preferably the gDNA is decreased in size to about 10 Kb on average. Morepreferably the gDNA is from 1 Kb to 50 Kb on average with from 5 Kb to15 Kb most preferred. In a most preferred embodiment the gDNA is atleast about 400 base pairs in length.

[0045] In a preferred embodiment the gDNA is substantially pure once itis immobilized on the solid support. Preferably the gDNA is purifiedprior to immobilization on the solid support. While it is not necessaryfor the gDNA to be 100% pure, in a preferred embodiment the gDNA is atleast 30% pure, with at least 50% pure being more preferred and at leastfrom 65% to 90% pure being most preferred.

[0046] In addition, the reactions using and reusing the sample materialas outlined herein may be accomplished in a variety of ways, as will beappreciated by those in the art. Components of the reaction may be addedsimultaneously, or sequentially, in any order, with preferredembodiments outlined below. In addition, the reaction may include avariety of other reagents which may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc., which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target.

[0047] In addition, in most embodiments, double stranded target nucleicacids are denatured to render them single stranded so as to permithybridization of the primers and other probes of the invention Apreferred embodiment utilizes a thermal step, generally by raising thetemperature of the reaction to about 95° C., although pH changes andother techniques may also be used.

[0048] In a preferred embodiment, the target nucleic acids or targetsequences are initially immobilized on a substrate. Attachment of thetarget sequences may be performed in a variety of ways, as will beappreciated by those in the art, including, but not limited to, chemicalor affinity capture (for example, including the incorporation attachmentmoieties such as derivatized nucleotides such as AminoLink orbiotinylated nucleotides that can then be used to attach the nucleicacid to a surface, as well as affinity capture by hybridization),cross-linking, and electrostatic attachment, etc. That is, an attachmentmoiety is attached to the target nucleic acid, i.e. genomic DNAsequences that allows for attachment to the substrate. By “attachmentmoiety” is meant a molecule or substance that mediates attachment of thegenomic DNA to the substrate. In a preferred embodiment, affinitycapture is used to attach the nucleic acids to the support. For example,nucleic acids can be derivatized, for example with one member of abinding pair, and the support derivatized with the other member, i.e. acomplementary member, of a binding pair. For example, the nucleic acidsmay be biotinylated (for example using enzymatic incorporation ofbiotinylated nucleotides, or by photoactivated cross-linking of biotin).Biotinylated nucleic acids can then be captured on streptavidin-coatedsurfaces, as is known in the art. In a preferred embodiment the targetnucleic acids are photobiotinylated with PHOTOPROBE™ Biotin Reagents(Vector Laboratories). In one embodiment the surfaces or supports arebeads to which the nucleic acids are attached. In a particularlypreferred embodiment the beads are magnetic beads.

[0049] Similarly, other hapten-receptor combinations can be used, suchas digoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthem be used to add the nucleic acid to the surface. Similarly, affinitycapture utilizing hybridization can be used to attach nucleic acids tosurface or bead. For example, a polyA tract can be attached bypolymerization with terminal transferase, or via ligation of an oligoAlinker, as is known in the art. This then allows for hybridization withan immobilized poly-T tract. Alternatively, chemical crosslinking may bedone, for example by photoactivated crosslinking of thymidine toreactive groups, as is known in the art. In a preferred embodiment thetarget nucleic acids are photobiotinylated

[0050] In some embodiments attachment of target nucleic acids isaccomplished by hybridizing the target nucleic acids to probes that areimmobilized on a solid support. That is, the immobilized probes serve toimmobilize the target nucleic acids.

[0051] Attachments can be covalent, although even relatively weakinteractions (i.e. non-covalent) can be sufficient to attach a nucleicacid to a surface, if there are multiple sites of attachment per eachnucleic acid. Thus, for example, electrostatic interactions can be usedfor attachment, for example by having beads carrying the opposite chargeto the bioactive agent.

[0052] A preferred embodiment utilizes covalent attachment of the targetsequences to a support. As is known in the art, there are a wide varietyof methods used to covalently attach nucleic acids to surfaces. Apreferred embodiment utilizes the incorporation of a chemical functionalgroup into the nucleic acid, followed by reaction with a derivatized oractivated surface. Examples include, but are not limited to AminoLink.

[0053] By “substrate”, “solid support”, “target substrate” or “targetsupport” or other grammatical equivalents herein is meant any materialto which a target nucleic acid can be attached. The target nucleic acidscan be attached either directly or indirectly as described herein. Aswill be appreciated by those in the art, the number of possiblesubstrates is very large. Possible substrates include inorganic andorganic substrates and are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, etc.), polysaccharides, cellulose,nylon or nitrocellulose, resins, silica or silica-based materialsincluding silicon and modified silicon, carbon, metals, inorganicglasses, plastics, and a variety of other polymers. Preferably thesubstrates include microfuge tubes, i.e. Eppendorf tubes. In a preferredembodiment the substrates include beads or microspheres. In oneembodiment the beads or microspheres are magnetic. In one embodiment thesubstrates are derivatized to accommodate attachment of the targetnucleic acids to the substrate.

[0054] The configuration of the target support is not crucial. What isimportant is that the target nucleic acids are immobilized to the targetsupport and can be manipulated. That is, the support should be amenableto a variety of reactions as described herein. While the substrate canbe flat (planar), other configurations of substrates may be used aswell; for example, target nucleic acids can be attached to beads ormicrospheres that can be deposited in reaction tubes or vessels orwells. That is, the target substrate may be microspheres to which thetarget nucleic acids are attached. The microspheres can then bedistributed on a surface. In some embodiments the surface containsreaction wells into which the beads are distributed, for examplemicrotiter plates as are known in the art and as described herein. Inone embodiment all of the beads to which an entire sample of targetnucleic acids are attached are deposited or distributed into a singlereaction well. Alternatively, a subpopulation of the beads to which asample of target nucleic acids are attached are deposited into differentwells. Generally, the subpopulation is not defined by a particularattribute, but is merely an aliquot of the complete sample of targetnucleic acids immobilized to beads. In one embodiment the microspheresare magnetic particles or microspheres. These magnetic particles orbeads can be immobilized with magnetic forces.

[0055] In one embodiment the target nucleic acids are immobilized intosample wells of a 96-well plate. Other formats of microtiter plates areknown in the art, i.e. 384 and 1536-well plates, and find use in theinvention. Alternatively, when target nucleic acids are attached tobeads, the beads are distributed into sample wells of the microtiterplate. The use of microtiter plates finds particular use in the methodsof the invention in that multiple target samples can be simultaneouslyprocessed. In the method of this invention, the multiple samples can besimultaneously processed sequentially. That is, as described herein, theinvention provides methods of reusing target nucleic acids. Whenmultiple samples of target nucleic acids are distributed in differentsample wells of a microtiter plate, the method provides for thesequential use of a plurality of target nucleic acids simultaneously.

[0056] Once the genomic DNA is applied to or immobilized on the surface,the target sequences are contacted with probes for analyses, includingdetection or genotyping, of the target sequences. Accordingly, asoutlined herein, the invention provides a number of different primersand probes. Probes and primers of the present invention are designed tohave at least a portion be complementary to a target sequence (eitherthe target sequence of the sample or to other probe sequences, such asportions of amplicons, as is described below), such that hybridizationof the target sequence and the probes of the present invention occurs.As outlined below, this complementarity need not be perfect; there maybe any number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions, andpreferably give the required specificity.

[0057] A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthand pH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 M sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of helix destabilizing agents such asformamide. The hybridization conditions may also vary when a non-ionicbackbone, i.e. PNA is used, as is known in the art. In addition,cross-linking agents may be added after target binding to cross-link,i.e. covalently attach, the two strands of the hybridization complex.

[0058] Thus, the assays are generally run under stringency conditionswhich allows formation of the first hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration, pH, organic solvent concentration, etc.

[0059] These parameters may also be used to control non-specificbinding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus itmay be desirable to perform certain steps at higher stringencyconditions to reduce non-specific binding.

[0060] The size of the primer and probe nucleic acids may vary, as willbe appreciated by those in the art, with each portion of the probe andthe total length of the probe in general varying from 5 to 500nucleotides in length. Each portion is preferably between 10 and 100being preferred, between 15 and 50 being particularly preferred, andfrom 10 to 35 being especially preferred, depending on the use andamplification technique. Thus, for example, the priming sites (includinguniversal priming sites) of the probes are each preferably about 15-20nucleotides in length, with 18 being especially preferred. The adaptersequences of the probes are preferably from 15-25 nucleotides in length,with 20 being especially preferred. The target specific portion of theprobe is preferably from 15-50 nucleotides in length.

[0061] In some embodiments probes include priming sites forpre-amplification reactions as described below. In this embodiment, theprobes include a pre-amplification priming site that is preferablypositioned 5′ to the universal priming site. In a preferred embodimentthe pre-amplification priming site is a priming site for RNA Polymerase.Preferably the priming site is a T3 or SP6 primer. In a preferredembodiment the priming site is includes a T7 RNA primer.

[0062] Accordingly, the present invention provides first target probesets. By “probe set” herein is meant a plurality of target probes thatare used in a particular multiplexed assay. In this context, pluralitymeans at least two, with more than 10 being preferred. More preferablythe probe set includes at least 100 probes with at least 1000 probesbeing more preferred. In one embodiment the number of probes is at least100 but less than 1,000,000 but is more preferably at least 200 and lessthan 5000. The number of probes will vary depending on the assay, sampleand purpose of the test. In one embodiment the number of probes is equalto the number of genes in the genome of a particular organism to beanalyzed. In some embodiments the number of probes is from one to threeprobes per locus or SNP to be identified. In addition, because there aregenerally multiple SNPs per gene, the probe sets include multiple probesper gene. That is, the probe sets include a plurality of probes for theidentification or detection of a gene.

[0063] Accordingly, the invention also provides for methods ofmultiplexed analysis of target nucleic acids. by “multiplex” is meantthe multiple analyses of a target nucleic acid. In one embodiment atleast 10 analyses are performed on target nucleic acids simultaneously.In a more preferred embodiment at least 50 simultaneous analyses areperformed, with at least 100 simultaneous analyses being performed. Inaddition, the invention provides for simultaneous analyses of at least1000 target nucleic acids.

[0064] The target probes comprise a first target specific sequence. By“target specific” sequence is meant sequence in a probe that issubstantially complementary to the target sequence. As outlined below,ligation probes each comprise a target-specific sequence. As will beappreciated by those in the art, the target-specific sequence comprisesa portion that will hybridize to all or part of the target sequence andincludes one or more particular single nucleotide polymorphisms (SNPs).As described above, the target specific sequence of the probe preferablyare from 15-50 nucleotides in length with from 20-35 being morepreferred.

[0065] In addition, the target probes comprise priming sites. By“priming sites” herein is meant a portion of the probe to which primershybridize to form amplification templates. By amplification templates ismeant a probe to which at least one amplification primer is hybridized.

[0066] Preferably the priming sites are universal priming sites. By“universal priming site” herein is meant a sequence of the probe thatwill bind a PCR primer for amplification. That is, all probes cancontain the same universal priming site or sequence, or subsets ofprobes can contain the same the same universal priming site or sequencewithin the subset, but the priming site or sequence is distinct fordifferent subsets. Accordingly, different subsets of probes containingthe same priming sites are used for multiplexing or detecting aplurality of target sequences. That is, for example, one set of probesincludes the same universal priming site, but multiple different targetspecific sequences. A second set of probes includes a different primingsite from the first set, but the probes of the second set contain thesame priming site. The probes of the second set also contain multipledifferent target specific sequences. As is appreciated by the skilledartisan, any number of sets can be used. In a preferred embodiment eachset comprises at least 5 different target specific sequences, morepreferably at least 50 different target specific sequences, with atleast 100 different target specific sequences being even more preferredand up to 1,000,000 different target sequences being most preferred.That is, in some embodiments each probe set includes from 100 to 1000different target sequences while in a most preferred embodiment eachprobe set includes from 100 to 10,000 different target sequences.

[0067] Each probe preferably comprises an upstream universal primingsite (UUP) and a downstream universal priming site (DUP). Again,“upstream” and “downstream” are not meant to convey a particular 5′-3′orientation, and will depend on the orientation of the system.Preferably, only a single UUP sequence and a single DUP sequence is usedin a probe set, although as will be appreciated by those in the art,different assays or different multiplexing analysis may utilize aplurality of universal priming sequences. In addition, the universalpriming sites are preferably located at the 5′ and 3′ termini of thetarget probe (or the ligated probe), as only sequences flanked bypriming sequences will be amplified. In some embodiments, for example,in the case of rolling circle embodiments, there may be a singleuniversal priming site.

[0068] In addition, universal priming sequences are generally chosen tobe as unique as possible given the particular assays and host genomes toensure specificity of the assay. In general, universal priming sequencesrange in size from about 5 to about 25 basepairs, with from about 10 toabout 20 being particularly preferred.

[0069] As will be appreciated by those in the art, the orientation ofthe two priming sites is different. That is, one PCR primer willdirectly hybridize to the first priming site, while the other PCR primerwill hybridize to the complement of the second priming site. Stateddifferently, the first priming site is in sense orientation, and thesecond priming site is in antisense orientation.

[0070] In a preferred embodiment the invention is directed to methods ofdetecting target sequences that comprise one or more positions for whichsequence information is desired, generally referred to herein as the“detection position” or “detection locus”. In a preferred embodiment,the detection position is a single nucleotide (sometimes referred to asa single nucleotide polymorphism (SNP)), although in some embodiments,it may comprise a plurality of nucleotides, either contiguous with eachother or separated by one or more nucleotides. By “plurality” as usedherein is meant at least two. As used herein, the base of a probe (e.g.the target probe) which basepairs with a detection position base in ahybrid is termed a “readout position” or an “interrogation position”.Thus, the target sequence comprises a detection position and the targetprobe comprises a readout position. In general, this embodiment utilizesthe OLA or RCA assay, as described below.

[0071] Thus, in a preferred embodiment, genotyping reactions areperformed on the immobilized DNA. These genotyping techniques fall intofour general categories: (1) techniques that rely on traditionalhybridization methods that utilize the variation of stringencyconditions (temperature, buffer conditions, etc.) to distinguishnucleotides at the detection position; (2) extension techniques that adda base (“the readout base”) to basepair with the nucleotide at thedetection position; (3) ligation techniques, that rely on thespecificity of ligase enzymes (or, in some cases, on the specificity ofchemical techniques), such that ligation reactions occur preferentiallyif perfect complementarity exists at the detection position; (4)cleavage techniques that also rely on enzymatic or chemical specificitysuch that cleavage occurs preferentially if perfect complementarityexists and (5) techniques that combine these methods.

[0072] As will be appreciated by those in the art, the reactionsdescribed below can take on a wide variety of formats. In oneembodiment, genomic DNA is attached to a solid support, and probescomprising priming sites are added to form hybridization complexes, in avariety of formats as outlined herein. The non-hybridized probes arethen removed, and the hybridization complexes are denatured. Thisreleases the probes (which frequently have been altered in some way).The probes are then amplified and detected, for example upon addition toan array of capture probes. Several embodiments of this have beendescribed above. Alternatively, genomic DNA is attached to a solidsupport, and genotyping reactions are done in formats that can allowamplification as well, either during the genotyping reaction (e.g.through the use of heat cycling) or after, without the use of universalprimers. Thus, for example, when labeled probes are used, they can behybridized to the immobilized genomic DNA, unbound materials removed,and then eluted and collected to be added to arrays. This may berepeated for amplification purposes, with the elution fractions pooledand added to the array. In addition, alternative amplification schemessuch as extending a product of the invasive cleavage reaction (describedbelow) to include universal primers or universal primers and adapterscan be performed. In one embodiment this allows the reuse of immobilizedtarget sequences with a different set or sets of target probes.

[0073] Simple Hybridization Detection or Genotyping

[0074] In a preferred embodiment, straight hybridization methods areused to elucidate the identity of the base at the detection position.Generally speaking, these techniques break down into two basic types ofreactions: those that rely on competitive hybridization techniques, andthose that discriminate using stringency parameters and combinationsthereof. Competitive hybridization In a preferred embodiment, the use ofcompetitive hybridization probes (generally referred to herein as“readout probes”) is performed to elucidate either the identity of thenucleotide(s) at the detection position or the presence of a mismatch.Readout probes also are known as target probes or target readout probesas described herein. For example, sequencing by hybridization has beendescribed (Drmanac et al., Genomics 4:114 (1989); Koster et al., NatureBiotechnology 14:1123 (1996); U.S. Pat. Nos. 5,525,464; 5,202,231 and5,695,940, among others, all of which are hereby expressly incorporatedby reference in their entirety).

[0075] It should be noted in this context that “mismatch” is a relativeterm and meant to indicate a difference in the identity of a base at aparticular position, termed the “detection position” herein, between twosequences. In general, sequences that differ from wild type sequencesare referred to as mismatches. However, particularly in the case ofSNPS, what constitutes “wild type” may be difficult to determine asmultiple alleles can be relatively frequently observed in thepopulation, and thus “mismatch” in this context requires the artificialadoption of one sequence as a standard. Thus, for the purposes of thisinvention, sequences are referred to herein as “match” and “mismatch”.Thus, the present invention may be used to detect substitutions,insertions or deletions as compared to a wild-type sequence. In general,probes of the present invention are designed to be complementary to atarget sequence, such that hybridization of the target and the probes ofthe present invention occurs. This complementarity need not be perfect;there may be any number of base pair mismatches that will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under the selected reaction conditions.

[0076] In a preferred embodiment, a plurality of probes (sometimesreferred to herein as “readout probes”, “detection probes”, “targetprobes” or “target readout probes”) are used to identify the base at thedetection position. In this embodiment, each different readout probecomprises a different detection label (which, as outlined below, can beeither a primary label or a secondary label) and a different base at theposition that will hybridize to the detection position of the targetsequence (herein referred to as the readout position) such thatdifferential hybridization will occur (see FIG. 1) That is, all otherparameters being equal, a perfectly complementary readout probe (a“match probe”) will in general be more stable and have a slower off ratethan a probe comprising a mismatch (a “mismatch probe”) at anyparticular temperature. Accordingly, by using different readout probes,each with a different base at the readout position and each with adifferent label, the identification of the base at the detectionposition is elucidated.

[0077] In one embodiment the readout probes are the same length.Preferably the probes have a similar melting temperature (Tm), althoughthis is not required. In an alternative embodiment, readout probes asdescribed herein need not be of the same length. That is, readout probescan be of different lengths. Using readout probes of different lengthsprovides the advantage that in varying the length of the probes, the Tmof the probes can be adjusted. This is beneficial in allowing uniformassay conditions can be used.

[0078] In one embodiment the readout probes comprise a detection label.By “detection label” or “detectable label” herein is meant a moiety thatallows detection. This may be a primary label (which can be directlydetected) or a secondary label (which is indirectly detected).

[0079] A primary label is one that can be directly detected, such as afluorophore. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal labels; and c) colored or luminescent dyes.Preferred labels include chromophores or phosphors but are preferablyfluorescent dyes. Suitable dyes for use in the invention include, butare not limited to, fluorescent lanthanide complexes, including those ofEuropium and Terbium, fluorescein, rhodamine, tetramethylrhodamine,eosin, erythrosin, coumarin, methyl-coumarins, quantum dots (alsoreferred to as “nanocrystals”), pyrene, Malacite green, stilbene,Lucifer Yellow, Cascade Blue™, Cy dyes (Cy3, Cy5, etc.), Texas Red,phycoerythrin, Bodipy, Alexa dyes and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland, herebyexpressly incorporated by reference. In a preferred embodiment, thedetection label used for competitive hybridization is a primary label.

[0080] In a preferred embodiment, the detectable label is a secondarylabel. A secondary label is one that is indirectly detected; forexample, a secondary label can bind or react with a primary label fordetection, can act on an additional product to generate a primary label(e.g. enzymes), or may allow the separation of the compound comprisingthe secondary label from unlabeled materials, etc. Secondary labels findparticular use in systems requiring separation of labeled and unlabeledprobes, such as SBE, OLA, invasive cleavage, etc. reactions; inaddition, these techniques may be used with many of the other techniquesdescribed herein. Secondary labels include, but are not limited to, oneof a binding partner pair; chemically modifiable moieties; nucleaseinhibitors, enzymes such horseradish peroxidase, alkaline phosphatases,luciferases, etc.

[0081] In a preferred embodiment, the secondary label is a bindingpartner pair. For example, the label may be a hapten or antigen, whichwill bind its binding partner. For example, suitable binding partnerpairs include, but are not limited to: antigens (such as proteins(including peptides)) and antibodies (including fragments thereof (FAbs,etc.)); proteins and small molecules, including biotin/streptavidin anddigoxygenin and antibodies; enzymes and substrates or inhibitors; otherprotein-protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners, are also suitable binding pairs. Nucleicacid—nucleic acid binding proteins pairs are also useful. In general,the smaller of the pair is attached to the NTP (or the probe) forincorporation into the extension primer. Preferred binding partner pairsinclude, but are not limited to, biotin (or imino-biotin) andstreptavidin, digeoxinin and Abs, and Prolinx™ reagents (seewww.prolinxinc com/ie4/home.hmtl).

[0082] In a preferred embodiment, the binding partner pair comprises aprimary detection label (attached to the NTP and therefore to theextended primer) and an antibody that will specifically bind to theprimary detection label. By “specifically bind” herein is meant that thepartners bind with specificity sufficient to differentiate between thepair and other components or contaminants of the system. The bindingshould be sufficient to remain bound under the conditions of the assay,including wash steps to remove non-specific binding. In someembodiments, the dissociation constants of the pair will be less thanabout 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ beingpreferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ being particularlypreferred.

[0083] In addition, the secondary label can be a chemically modifiablemoiety. In this embodiment, labels comprising reactive functional groupsare incorporated into the nucleic acid. Subsequently, primary labels,also comprising functional groups, may be added to these reactivegroups. As is known in the art, this may be accomplished in a variety ofways. Preferred functional groups for attachment are amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the primarylabels can be attached using functional groups on the enzymes. Forexample, primary labels containing amino groups can be attached tosecondary labels comprising amino groups, for example using linkers asare known in the art; for example, homo-or hetero-bifunctional linkersas are well known (see 1994 Pierce Chemical Company catalog, technicalsection on cross-linkers, pages 155-200, incorporated herein byreference).

[0084] Accordingly, in some embodiments a detectable label isincorporated into the readout probe. In a preferred embodiment, a set ofreadout probes are used, each comprising a different base at the readoutposition. In some embodiments, each readout probe comprises a differentlabel, that is distinguishable from the others. For example, a firstlabel may be used for probes comprising adenosine at the readoutposition, a second label may be used for probes comprising guanine atthe readout position, etc. In a preferred embodiment, the length andsequence of each readout probe is identical except for the readoutposition, although this need not be true in all embodiments.

[0085] The number of readout probes used will vary depending on the enduse of the assay. For example, many SNPs are biallelic, and thus tworeadout probes, each comprising an interrogation base that will basepairwith one of the detection position bases. For sequencing, for example,for the discovery of SNPs, a set of four readout probes are used.

[0086] In one embodiment labels are not incorporated into the readoutprobes. Rather, the readout probes are used as a template for subsequentamplification reactions described in more detail below. Generally, inthis embodiment the readout probe includes priming sequences asdescribed herein and a label is incorporated into the amplificationproduct, i.e. the amplicon. As described above, the label may be eitherdirect or indirect.

[0087] Accordingly, the method includes adding target readout probes totarget nucleic acids that are immobilized on a surface. Unbound probesare washed away or removed from the complex. The bound probes are thenremoved, i.e. denatured, from the complex and collected. In a preferredembodiment, a second set of readout probes is then added to theimmobilized target nucleic acids. The immobilized target nucleic acidscan be reused indefinitely as described below. In one embodiment thereadout probes contain a label. In an alternative embodiment the readoutprobes contain priming sequences and are amplified. Labels areincorporated into the amplification products (amplicons). Finally, theidentity of the isolated probes or amplicons is determined by contactingthe probes with an array as described below.

[0088] Stringency Variation

[0089] In a preferred embodiment, sensitivity to variations instringency parameters are used to determine either the identity of thenucleotide(s) at the detection position or the presence of a mismatch.As a preliminary matter, the use of different stringency conditions suchas variations in temperature and buffer composition to determine thepresence or absence of mismatches in double stranded hybrids comprisinga single stranded target sequence and a probe is well known.

[0090] With particular regard to temperature, as is known in the art,differences in the number of hydrogen bonds as a function of basepairingbetween perfect matches and mismatches can be exploited as a result oftheir different Tms (the temperature at which 50% of the hybrid isdenatured). Accordingly, a hybrid comprising perfect complementaritywill melt at a higher temperature than one comprising at least onemismatch, all other parameters being equal. (It should be noted that forthe purposes of the discussion herein, all other parameters (i.e. lengthof the hybrid, nature of the backbone (i.e. naturally occuring ornucleic acid analog), the assay solution composition and the compositionof the bases, including G-C content are kept constant). However, as willbe appreciated by those in the art, these factors may be varied as well,and then taken into account.)

[0091] In general, as outlined herein, high stringency conditions arethose that result in perfect matches remaining in hybridizationcomplexes, while imperfect matches melt off. Similarly, low stringencyconditions are those that allow the formation of hybridization complexeswith both perfect and imperfect matches. High stringency conditions areknown in the art; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al., both of which are hereby incorporated byreference. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, “Overview of principles of hybridization and the strategy ofnucleic acid assays” (1993). Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH and nucleicacid concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 M sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide. Inanother embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

[0092] As will be appreciated by those in the art, mismatch detectionusing temperature may proceed in a variety of ways, and is similar tothe use of readout probes as outlined above. Again, as outlined above, aplurality of readout probes may be used in a sandwich format; in thisembodiment, all the probes may bind at permissive, low temperatures(temperatures below the Tm of the mismatch); however, repeating theassay at a higher temperature (above the Tm of the mismatch) only theperfectly matched probe may bind. Thus, this system may be run withreadout probes with different detectable labels, as outlined above.Alternatively, a single probe may be used to query whether a particularbase is present.

[0093] Similarly, variations in buffer composition may be used toelucidate the presence or absence of a mismatch at the detectionposition. Suitable conditions include, but are not limited to, formamideconcentration. Thus, for example, “low” or “permissive” stringencyconditions include formamide concentrations of 0 to 10%, while “high” or“stringent” conditions utilize formamide concentrations of ≦40%. Lowstringency conditions include NaCl concentrations of ≧1 M, and highstringency conditions include concentrations of ≦0.3 M. Furthermore, lowstringency conditions include MgCl₂ concentrations of ≧10 mM, moderatestringency as 1-10 mM, and high stringency conditions includeconcentrations of ≦1 mM.

[0094] In this embodiment, as for temperature, a plurality of readoutprobes may be used, with different bases in the readout position (andoptionally different labels). Running the assays under the permissiveconditions and repeating under stringent conditions will allow theelucidation of the base at the detection position.

[0095] Accordingly, the method includes adding target readout probes totarget nucleic acids that are immobilized on a surface. Complexes areformed under permissive conditions, followed by increasing thestringency which results in release of the mismatched probe. Unboundprobes are washed away or removed from the complex. The bound probes arethen removed, i.e. denatured, from the complex and collected. In apreferred embodiment, a second set of readout probes are then added tothe immobilized target nucleic acids. The target nucleic acids can bereused indefinitely as described below. In one embodiment the readoutprobes contain a label. In an alternative embodiment the readout probescontain priming sequences and are amplified. Labels are incorporatedinto the amplification products (amplicons). Finally, the identity ofthe isolated probes or amplicons is determined by contacting the probeswith an array as described below.

[0096] Extension Genotyping

[0097] In this embodiment, any number of techniques are used to add anucleotide to the readout position of an extension probe. By “extensionprobe” is meant a probe hybridized to the target sequence adjacent tothe detection position. Extension probes also are included in thedefinition of readout probes or target probes. By relying on enzymaticspecificity, preferentially a perfectly complementary base is added. Allof these methods rely on the enzymatic incorporation of nucleotides atthe detection position. This may be done using chain terminating dNTPs,such that only a single base is incorporated (e.g. single base extensionmethods), or under conditions that only a single type of nucleotide isadded followed by identification of the added nucleotide (extension andpyrosequencing techniques).

[0098] Single Base Extension

[0099] In one embodiment, single base extension (SBE; sometimes referredto as “minisequencing”) is used to determine the identity of the base atthe detection position. Briefly, SBE is a technique that utilizes anextension primer (also included withing the definition of readout probe)that hybridizes to the target nucleic acid immediately adjacent to thedetection position. A polymerase (generally a DNA polymerase) is used toextend the 3′ end of the primer with a nucleotide analog labeled adetection label as described herein. Based on the fidelity of theenzyme, a nucleotide is only incorporated into the readout position ofthe growing nucleic acid strand if it is perfectly complementary to thebase in the target strand at the detection position. The nucleotide maybe derivatized such that no further extensions can occur, so only asingle nucleotide is added. Once the labeled nucleotide is added,detection of the label proceeds as outlined herein. See generallySylvanen et al., Genomics 8:684-692 (1990); U.S. Pat. Nos. 5,846,710 and5,888,819; Pastinen et al., Genomics Res. 7(6):606-614 (1997); all ofwhich are expressly incorporated herein by reference.

[0100] The reaction is initiated by contacting the assay complexcomprising the immobilized target sequence (i.e. the array) to asolution comprising a first nucleotide. By “nucleotide” in this contextherein is meant a deoxynucleoside-triphosphate (also calleddeoxynucleotides or dNTPs, e.g. dATP, dTTP, dCTP and dGTP). In general,the nucleotides comprise a detectable label, which may be either aprimary or a secondary label. In addition, the nucleotides may benucleotide analogs, depending on the configuration of the system. Forexample, if the dNTPs are added in sequential reactions, such that onlya single type of dNTP can be added, the nucleotides need not be chainterminating. In addition, in this embodiment, the dNTPs may all comprisethe same type of label.

[0101] Alternatively, if the reaction comprises more than one dNTP, thedNTPs should be chain terminating, that is, they have a blocking orprotecting group at the 3′ position such that no further dNTPs may beadded by the enzyme. As will be appreciated by those in the art, anynumber of nucleotide analogs may be used, as long as a polymerase enzymewill still incorporate the nucleotide at the readout position. Preferredembodiments utilize dideoxy-triphosphate nucleotides (ddNTPs) andhalogenated dNTPs. Generally, a set of nucleotides comprising ddATP,ddCTP, ddGTP and ddTTP is used, each with a different detectable label,although as outlined herein, this may not be required.

[0102] In a preferred embodiment, the nucleotide analogs comprise adetectable label, which can be either a primary or secondary detectablelabel. Preferred primary labels are those outlined above forinterrogation labels. However, the enzymatic incorporation ofnucleotides comprising fluorophores is may be poor under manyconditions; accordingly, a preferred embodiment utilizes secondarydetectable labels. In addition, as outlined below, the use of secondarylabels may also facilitate the removal of unextended probes.

[0103] In addition, as will be appreciated by those in the art, thesingle base extension reactions of the present invention allow theprecise incorporation of modified bases into a growing nucleic acidstrand. Thus, any number of modified nucleotides may be incorporated forany number of reasons, including probing structure-functionrelationships (e.g. DNA:DNA or DNA:protein interactions), cleaving thenucleic acid, crosslinking the nucleic acid, incorporate mismatches,etc.

[0104] In addition to a first nucleotide, the solution also comprises anextension enzyme, generally a DNA polymerase. Suitable DNA polymerasesinclude, but are not limited to, the Klenow fragment of DNA polymeraseI, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymeraseand Phi29 DNA polymerase. If the NTP is complementary to the base of thedetection position of the target sequence, which is adjacent to theextension primer, the extension enzyme will add it to the extensionprimer at the readout position. Thus, the extension primer is modified,i.e. extended, to form a modified primer, sometimes referred to hereinas a “newly synthesized strand”. If desired, the temperature of thereaction can be adjusted (or cycled) such that amplification occurs,generating a plurality of modified primers.

[0105] In addition, since unextended primers do not comprise labels, theunextended primers need not be removed. However, they may be, ifdesired, as outlined below; for example, if a large excess of primersare used, there may not be sufficient signal from the extended primerscompeting for binding to the surface. In addition, removal of unextendedprimers is desirable when extended primers are to be amplified. That is,when amplification follows an extension reaction, it is desirable toremove the unextended primers so that they are also not amplified.

[0106] As will be appreciated by those in the art, the determination ofthe base at the detection position can proceed in several ways. In apreferred embodiment, the reaction is run with all four nucleotides(assuming all four nucleotides are required), each with a differentlabel, as is generally outlined herein. Alternatively, a single label isused, by using four reactions: this may be done either by using a singlesubstrate and sequential reactions, or by using a sample divided intofour reaction wells. For example, dATP can be added to the assaycomplex, and the generation of a signal evaluated; the dATP can beremoved and dTTP added, etc. Alternatively, four reaction wells can beused; the first well includes dATP, the second includes dTTP, etc., andthe presence or absence of a signal evaluated.

[0107] Alternatively, ratiometric analysis can be done; for example, twolabels, “A” and “B”, on two substrates (e.g. two reaction wells) can bedone. In this embodiment, two sets of primer extension reactions areperformed, each in two wells, with each reaction containing a completeset of four chain terminating NTPs. The first reaction contains two “A”labeled nucleotides and two “B” labeled nucleotides (for example, A andC may be “A” labeled, and G and T may be “B” labeled). The secondreaction also contains the two labels, but switched; for example, A andG are “A” labeled and T and C are “B” labeled. Following the reaction,the modified primers are removed from the immobilized target nucleicacid and analyzed on a bead array as described below. In a preferredembodiment the modified probes from each reaction are contacted orimmobilized on separate beads as described below and detected on a beadarray. This reaction allows a biallelic marker to be ratiometricallyscored; that is, the intensity of the two labels in two different“color” channels on a single bead is compared, using data from a set oftwo hybridized arrays. For instance, if the marker is A/G, then thefirst reaction on the first bead is used to calculate a ratiometricgenotyping score; if the marker is A/C, then the second reaction on thesecond bead is used for the calculation; if the marker is G/T, then thesecond bead is used, etc. This concept can be applied to all possiblebiallelic marker combinations “Scoring” a genotype using a single beadratiometric score allows a much more robust genotyping than scoring agenotype using a comparison of absolute or normalized intensitiesbetween two different arrays.

[0108] As will be appreciated by the skilled artisan, while SBE is apowerful technique for determining the nucleotide at the detectionposition of a target nucleic acid, the labeled target probe can notamplified with universal primers in this configuration. That is, whilespecific primers can be used to amplify the modified target probe,universal primers are not as there are not primers that are upstream anddownstream of the detection position. When specific primers are used, alabel can be incorporated into the amplicon as described herein. Inaddition, the product of the reaction can be “pre-amplified” asdescribed herein. Thus, by reusing the original sample DNA, samplepreparation for this and other assays is greatly simplified.

[0109] Although straight SBE is not amenable to amplification withuniversal primers, a configuration that does allow for amplificationwith universal primers is SBE followed by oligonucleotide ligation assay(OLA), i.e. the Genetic Bit Assay. OLA is described in more detailbelow. In this embodiment, an extension probe is hybridized to thetarget nucleic acids and the SBE reaction is performed as describedabove. Extension probe also is included in the definition of targetprobe as defined herein. In addition, an upstream ligation probe ishybridized to the target nucleic acid. The upstream ligation probehybridizes to the target nucleic acid immediately adjacent to thedetection position. Following incorporation of the appropriatenucleotide into the extension probe as a result of the SBE reaction, thecomplex is contacted with a ligase that only ligates the modifiedextension probe and upstream ligation probe when the correct nucleotidehas been incorporated into the extension probe. Following ligation, theligated probe is removed from the target nucleic acid and eitherdirectly detected on an array as described below, or amplified. When theligated probe is to be amplified, preferably the target probes, i.e.either the upstream ligation primer or extension primer contain primingsites for amplification primers. In a preferred embodiment the primingsites are universal priming sties as described herein.

[0110] Once the assay is complete and the extended probe (when SBE isused) or the ligated probe (when Genetic bit is used) are removed fromthe target nucleic acid, the target nucleic acid can be reused asdescribed herein. That is, the immobilized target nucleic acid can becontacted with a subsequent set of probes for additional analyses.

[0111] Allelic PCR

[0112] In a preferred embodiment, the method used to detect the base atthe detection position is allelic PCR, referred to herein as “aPCR”. Asdescribed in Newton et al., Nucl. Acid Res. 17:2503 (1989), herebyexpressly incorporated by reference, allelic PCR allows single basediscrimination based on the fact that the PCR reaction does not proceedwell if the terminal 3′-nucleotide is mismatched, assuming the DNApolymerase being used lacks a 3′-exonuclease proofreading activity.Accordingly, the identification of the base proceeds by using allelicPCR primers (sometimes referred to herein as aPCR primers) that havereadout positions at their 3′ ends. aPCR primers also are includedwithin the definition of readout probes or target probes. Thus thetarget sequence comprises a first domain comprising at its 5′ end adetection position.

[0113] In general, aPCR may be briefly described as follows. A targetnucleic acid is immobilized on a substrate as described herein. Thetarget nucleic acid is then denatured, generally by raising thetemperature, and then cooled in the presence of an excess of a aPCRprimer, which then hybridizes to the first target strand. If the readoutposition of the aPCR primer basepairs correctly with the detectionposition of the target sequence, a DNA polymerase (again, that lacks3′-exonuclease activity) then acts to extend the primer with dNTPs,resulting in the synthesis of a new strand forming a hybridizationcomplex. The sample is then heated again, to disassociate thehybridization complex, and the process is repeated. By using a secondPCR primer for the complementary target strand, rapid and exponentialamplification occurs. In one embodiment the dissociated extended primeris removed from the immobilized target nucleic acid prior to subsequentamplification cycles, although this is not required. When the extendedprimer is removed from the immobilized target the target can be reusedwith a different set of primers. Alternatively, a different assay can beperformed on the same target nucleic acid. Thus aPCR steps aredenaturation, annealing and extension. The particulars of aPCR are wellknown, and include the use of a thermostable polymerase such as Taq Ipolymerase and thermal cycling.

[0114] Accordingly, the aPCR reaction requires at least one aPCR primer,a polymerase, and a set of dNTPs. As outlined herein, the primers maycomprise the label, or one or more of the dNTPs may comprise a label. Asdescribed above for SBE, when only one aPCR primer is used,amplification with universal primers will not occur. However, when two aPCR primers are used, each of the primers contains a priming siteupstream of the target sequence. During each cycle of aPCR, theresulting extended product contains not only the amplified targetsequence, but also the sequence of each of the primers flanking thetarget specific sequences. As such, universal primers can be used toamplify the product.

[0115] It should be noted in this embodiment, at least one cycle ofamplification with two aPCR primers is required in order to generate aproduct that contains two priming sites. Again, preferably the primingsites are universal priming sites.

[0116] Furthermore, the aPCR reaction may be run as a competition assayof sorts. For example, for biallelic SNPS, a first aPCR primercomprising a first base at the readout position and a first label, and asecond aPCR primer comprising a different base at the readout positionand a second label, may be used. The PCR primer for the other strand isthe same. The examination of the ratio of the two colors can serve toidentify the base at the detection position.

[0117] The amplicons produced by the aPCR reaction and subsequentuniversal amplification steps can be analyzed on an array as describedbelow. In addition, the immobilized target nucleic acid can be reused ina subsequent aPCR reaction or different detection or genotyping reactionas described herein.

[0118] Ligation Techniques for Genotyping

[0119] In this embodiment, the readout of the base at the detectionposition proceeds using a ligase. In this embodiment, it is thespecificity of the ligase which is the basis of the genotyping; that is,ligases generally require that the 5′ and 3′ ends of the ligation probeshave perfect complementarity to the target for ligation to occur.Ligation probes also are included within the definition of readoutprobes or target probes.

[0120] In a preferred embodiment, the identity of the base at thedetection position proceeds utilizing the OLA. The method can be run atleast two different ways; in a first embodiment, only one strand of atarget sequence is used as a template for ligation; alternatively, bothstrands may be used; the latter is generally referred to as LigationChain Reaction or LCR. See generally U.S. Pat. Nos. 5,185,243 and5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO90/01069; WO 89/12696; and WO 89/09835, and U.S. S. Nos. 60/078,102 and60/073,011, all of which are incorporated by reference.

[0121] This method is based on the fact that two probes can bepreferentially ligated together, if they are hybridized to a targetstrand and if perfect complementarity exists at the two bases beingligated together. Thus, in this embodiment, the immobilized targetsequence comprises a contiguous first target domain comprising thedetection position and a second target domain adjacent to the detectionposition. That is, the detection position is “between” the rest of thefirst target domain and the second target domain. A first ligation probeis hybridized to the first target domain and a second ligation probe ishybridized to the second target domain. If the first ligation probe hasa base perfectly complementary to the detection position base, and theadjacent base on the second probe has perfect complementarity to itsposition, a ligation structure is formed such that the two probes can beligated together to form a ligated probe. If this complementarity doesnot exist, no ligation structure is formed and the probes are notligated together to an appreciable degree. In addition, as is more fullyoutlined herein, this method may also be done using ligation probes thatare separated by one or more nucleotides, if dNTPs and a polymerase areadded (this is sometimes referred to as “Genetic Bit” analysis).

[0122] In a preferred embodiment, LCR is done for two strands of adouble-stranded target sequence. The immobilized target sequence isdenatured, and two sets of probes are added: one set as outlined abovefor one strand of the target, and a separate set (i.e. third and fourthprimer probe nucleic acids) for the other strand of the target, both ofwhich are part of a particular target set.

[0123] As will be appreciated by those in the art, the ligation productcan be detected in a variety of ways.

[0124] Preferably, detection is accomplished by removing the unligatedlabeled probe from the reaction. Preferably, after the OLA assay isperformed, the unligated oligonucleotides are removed by washing underappropriate stringency to remove unligated oligonucleotides. In oneembodiment, one of the probes comprises a label, which is detected on anarray as described herein. In a preferred embodiment the ligationproduct is amplified to produce an amplicon; preferably a label isincorporated into the amplicon as described herein. In a preferredembodiment the ligation probes comprise priming sites for amplificationas described herein. That is, flaking the target specific sequence ofboth of the ligation probes the probes include priming sequences. Thepriming sequences allow for amplification of the ligated probe. In apreferred embodiment the priming sequences are universal primingsequences as described herein.

[0125] In one embodiment, once the ligated probe is removed from theimmobilized target nucleic acid, the target nucleic acid is reused in asubsequent analysis, i.e. detection or genotyping assay, as describedherein. The ligated probe, or amplicon resulting from the amplificationreaction, is detected on an array as described below.

[0126] Padlock Probe Ligation

[0127] In a preferred embodiment, the ligation probes are specializedprobes called “padlock probes” (which also are within the definition ofreadout probes) Nilsson et al, 1994, Science 265:2085. These probes havea first ligation domain that is identical to a first ligation probe, inthat it hybridizes to a first target sequence domain, and a secondligation domain, identical to the second ligation probe, that hybridizesto an adjacent target sequence domain. Again, as for OLA, the detectionposition can be either at the 3′ end of the first ligation domain or atthe 5′ end of the second ligation domain. However, the two ligationdomains are connected by a linker, frequently nucleic acid. Theconfiguration of the system is such that upon ligation of the first andsecond ligation domains of the padlock probe, the probe forms a circularprobe, and forms a complex with the target sequence wherein the targetsequence is “inserted” into the loop of the circle.

[0128] In this embodiment, the unligated probes may be removed throughdegradation (for example, through a nuclease), as there are no “freeends” in the ligated probe.

[0129] In a preferred embodiment the “padlock probe” contains a primingsite for amplification. Preferably the priming site is located in the“linker” region of the padlock probe. Only one priming site is requiredas amplification proceeds around the closed circle resulting in a singleamplicon containing multiple copies of the probe sequence. In apreferred embodiment the padlock probe contains with its sequence arestriction site. As such, following amplification of the probesequence, the amplicon can be cleaved resulting in multiple segments ofamplicon DNA containing the sequence of the padlock probe. Followingcleavage, the cleaved amplicon products can be detected on an array asdescribed herein.

[0130] Following removal of the padlock probe from the immobilizedtarget nucleic acid, the immobilized target nucleic acid can be used forsubsequent analyses including detection and genotyping analysis.

[0131] Cleavage Techniques for Genotyping

[0132] In a preferred embodiment, the specificity for genotyping isprovided by a cleavage enzyme There are a variety of enzymes known tocleave at specific sites, either based on sequence specificity, such asrestriction endonucleases, or using structural specificity, such as isdone through the use of invasive cleavage technology.

[0133] Endonuclease Techniques

[0134] In a preferred embodiment, enzymes that rely on sequencespecificity are used. In general, these systems rely on the cleavage ofdouble stranded sequence containing a specific sequence recognized by anuclease, preferably an endonuclease including resolvases.

[0135] These systems may work in a variety of ways, as outlined in U.S.Ser. No. 09/556,463, filed Apr. 21, 2000, which is expresslyincorporated herein by reference. In one embodiment, a labeled readoutprobe is used; the binding of the target sequence forms a doublestranded sequence that a restriction endonuclease can then recognize andcleave, if the correct sequence is present. The cleavage results in theloss of the label, and thus a loss of signal.

[0136] Alternatively, as will be appreciated by those in the art, alabelled target sequence may be used as well; for example, a labelledprimer may be used in the PCR amplification of the target, such that thelabel is incorporated in such a manner as to be cleaved off by theenzyme.

[0137] Alternatively, the readout probe (or, again, the target sequence)may comprise both a fluorescent label and a quencher, as is known in theart. In this embodiment, the label and the quencher are attached todifferent nucleosides, yet are close enough that the quencher moleculeresults in little or no signal being present. Upon the introduction ofthe enzyme, the quencher is cleaved off, leaving the label, and allowingsignalling by the label.

[0138] In addition, as will be appreciated by those in the art, thesesystems can be both solution-based assays or solid-phase assays, asoutlined herein.

[0139] Furthermore, there are some systems that do not require cleavagefor detection; for example, some nucleic acid binding proteins will bindto specific sequences and can thus serve as a secondary label. Forexample, some transcription factors will bind in a highly sequencedependent manner, and can distinguish between two SNPs. Having bound tothe hybridization complex, a detectable binding partner can be added fordetection. In addition, mismatch binding proteins based on mutatedtranscription factors can be used.

[0140] In addition, as will be appreciated by those in the art, thistype of approach works with other cleavage methods as well, for examplethe use of invasive cleavage methods, as outlined below.

[0141] Invasive Cleavage

[0142] In a preferred embodiment, the determination of the identity ofthe base at the detection position of the target sequence proceeds usinginvasive cleavage technology. As outlined above for amplification,invasive cleavage techniques rely on the use of structure-specificnucleases, where the structure can be formed as a result of the presenceor absence of a mismatch Generally, invasive cleavage technology may bedescribed as follows. A target nucleic acid is recognized by twodistinct probes. A first probe, generally referred to herein as an“invader” probe, is substantially complementary to a first portion ofthe target nucleic acid. A second probe, generally referred to herein asa “signal probe”, is partially complementary to the target nucleic acid;the 3′ end of the signal oligonucleotide is substantially complementaryto the target sequence while the 5′ end is non-complementary andpreferably forms a single-stranded “tail” or “arm”. Thenon-complementary end of the second probe preferably comprises a“generic” or “unique” sequence, frequently referred to herein as a“detection sequence”, that is used to indicate the presence or absenceof the target nucleic acid, as described below. The detection sequenceof the second probe preferably comprises at least one detectable label.Alternative methods have the detection sequence functioning as a targetsequence for a capture probe, and thus rely on sandwich configurationsusing label probes.

[0143] Hybridization of the first and second oligonucleotides near oradjacent to one another on the target nucleic acid forms a number ofstructures. In a preferred embodiment, a forked cleavage structure formsand is a substrate of a nuclease which cleaves the detection sequencefrom the signal oligonucleotide. The site of cleavage is controlled bythe distance or overlap between the 3′ end of the invaderoligonucleotide and the downstream fork of the signal oligonucleotide.Therefore, neither oligonucleotide is subject to cleavage whenmisaligned or when unattached to target nucleic acid.

[0144] As above, the invasive cleavage assay is preferably performed onan array format. In a preferred embodiment, the signal probe has adetectable label, attached 5′ from the site of nuclease cleavage (e.g.within the detection sequence) and a capture tag, as described hereinfor removal of the unreacted products (e.g. biotin or other hapten) 3′from the site of nuclease cleavage. After the assay is carried out, theuncleaved probe and the 3′ portion of the cleaved signal probe (e.g. thethe detection sequence) may be extracted, for example, by binding tostreptavidin beads or by crosslinking through the capture tag to produceaggregates or by antibody to an attached hapten. By “capture tag” hereinis a meant one of a pair of binding partners as described above, such asantigen/antibody pairs, digoxygenenin, dinitrophenol, etc.

[0145] The cleaved 5′ region, e.g. the detection sequence, of the signalprobe, comprises a label and is detected and optionally quantitated. Inone embodiment, the cleaved 5′ region is hybridized to a probe on anarray (capture probe) and optically detected. As described below, manydifferent signal probes can be analyzed in parallel by hybridization totheir complementary probes in an array. In a preferred embodiment,combination techniques are used to obtain higher specificity and reducethe detection of contaminating uncleaved signal probe or incorrectlycleaved product, an enzymatic recognition step is introduced in thearray capture procedure. For example, as more fully outlined below, thecleaved signal probe binds to a capture probe to produce adouble-stranded nucleic acid in the array. In this embodiment, the 3′end of the cleaved signal probe is adjacent to the 5′ end of one strandof the capture probe, thereby, forming a substrate for DNA ligase(Broude et al. 1991. PNAS 91: 3072-3076). Only correctly cleaved productis ligated to the capture probe. Other incorrectly hybridized andnon-cleaved signal probes are removed, for example, by heatdenaturation, high stringency washes, and other methods that disruptbase pairing.

[0146] Accordingly, the present invention provides methods ofdetermining the identity of a base at the detection position of a targetsequence. In this embodiment, the target sequence comprises, 5′ to 3′, afirst target domain comprising an overlap domain comprising at least anucleotide in the detection position, and a second target domaincontiguous with the detection position. A first probe (the “invaderprobe”) is hybridized to the first target domain of the target sequence.A second probe (the “signal probe”), comprising a first portion thathybridizes to the second target domain of the target sequence and asecond portion that does not hybridize to the target sequence, ishybridized to the second target domain. If the second probe comprises abase that is perfectly complementary to the detection position acleavage structure is formed. The addition of a cleavage enzyme, such asis described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,029;5,541,311 and 5,843,669, all of which are expressly incorporated byreference, results in the cleavage of the detection sequence from thesignalling probe. This then can be used as a target sequence in an assaycomplex.

[0147] In addition, as for a variety of the techniques outlined herein,unreacted probes (i.e. signaling probes, in the case of invasivecleavage), may be removed using any number of techniques. For example,the use of a binding partner coupled to a solid support comprising theother member of the binding pair can be done. Similarly, after cleavageof the primary signal probe, the newly created cleavage products can beselectively labeled at the 3′ or 5′ ends using enzymatic or chemicalmethods.

[0148] Again, as outlined above, the detection of the invasive cleavagereaction can occur directly, in the case where the detection sequencecomprises at least one label, or indirectly, using sandwich assays,through the use of additional probes; that is, the detection sequencescan serve as target sequences, and detection may utilize amplificationprobes, capture probes, capture extender probes, label probes, and labelextender probes, etc.

[0149] In addition, as for most of the techniques outlined herein, thesetechniques may be done for the two strands of a double-stranded targetsequence. The target sequence is denatured, and two sets of probes areadded: one set as outlined above for one strand of the target, and aseparate set for the other strand of the target.

[0150] Thus, the invasive cleavage reaction requires, in no particularorder, an invader probe, a signalling probe, and a cleavage enzyme.

[0151] As for other methods outlined herein, the invasive cleavagereaction may be done as a solution based assay or a solid phase assay.

[0152] Solution-Based Invasive Cleavage

[0153] The invasive cleavage reaction may be done in solution, followedby addition of one of the components to an array, with optional (butpreferable) removal of unreacted probes. For example, the reaction iscarried out in solution, using a capture tag (i.e. a member of a bindingpartner pair) that is separated from the label on the detection sequencewith the cleavage site. After cleavage (dependent on the base at thedetection position), the signaling probe is cleaved. The capture tag isused to remove the uncleaved probes (for example, using magneticparticles comprising the other member of the binding pair), and theremaining solution is added to the array. In this embodiment, thedetection sequence can effectively act as an adapter sequence. Inalternate embodiments, the detection sequence is unlabelled and anadditional label probe is used; as outlined below, this can be ligatedto the hybridization complex.

[0154] Solid-Phase Based Assays

[0155] The invasive cleavage reaction can also be done as a solid-phaseassay. The target sequence can be attached to the array using a captureprobe (in addition, although not shown, the target sequence may bedirectly attached to the array). In a preferred embodiment, thesignalling probe comprises both a fluorophore label (attached to theportion of the signalling probe that hybridizes to the target) and aquencher (generally on the detection sequence), with a cleavage site inbetween. Thus, in the absence of cleavage, very little signal is seendue to the quenching reaction. After cleavage, however, the detectionsequence is removed, along with the quencher, leaving the unquenchedfluorophore. Similarly, the invasive probe may be attached to the array.

[0156] In a preferred embodiment, the invasive cleavage reaction isconfigured to utilize a fluorophorequencher reaction. A signaling probecomprising both a fluorophore and a quencher is attached to the bead.The fluorophore is contained on the portion of the signaling probe thathybridizes to the target sequence, and the quencher is contained on aportion of the signaling probe that is on the other side of the cleavagesite (termed the “detection sequence” herein). In a preferredembodiment, it is the 3′ end of the signaling probe that is attached tothe bead (although as will be appreciated by those in the art, thesystem can be configured in a variety of different ways, includingmethods that would result in a loss of signal upon cleavage). Thus, thequencher molecule is located 5′ to the cleavage site. Upon assembly ofan assay complex, comprising the target sequence, an invader probe, anda signalling probe, and the introduction of the cleavage enzyme, thecleavage of the complex results in the disassociation of the quencherfrom the complex, resulting in an increase in fluorescence.

[0157] In this embodiment, suitable fluorophore-quencher pairs are asknown in the art. For example, suitable quencher molecules compriseDabcyl.

[0158] Pre-Amplification of Detection or Genotyping Products

[0159] In one embodiment, the probes include a pre-amplification primingsite. The pre-amplification priming sites contain sites to which primersfor DNA or RNA polymerases will hybridize. As described above, DNApolymerases or RNA polymerases can be used, although RNA polymerases arepreferred.

[0160] When RNA polymerases are used, the pre-amplification primingsites include a priming site for the RNA polymerase. Preferred primersequences include but are not limited to SP6, T3 or T7 primers. T7 isthe preferred primer.

[0161] In one embodiment, the pre-amplification priming site ispositioned upstream of the upstream priming site or UUP when universalpriming sites are used for amplification. In addition, only onepre-amplification priming site is necessary. That is, for example, whenSBE is used, only one pre-amplification priming site need be included inthe target probe. When OLA is used, which requires two ligation probes,only one of the ligation probes need contain a pre-amplification primingsite, although both ligation probes can contain one. When both ligationprobes contain one, the pre-amplification priming site in the downstreampriming site should be positioned downstream of the downstream primingsite. That is, when two pre-amplification priming sites are used, theyshould be positioned such that they flank both the target specificsequence and the upstream and downstream priming sites. As such, uponcomplection of a pre-amplification reaction, all of the interveningsequences, including the priming sites are amplified.

[0162] Amplification

[0163] Following either the initial detection or genotyping reaction orthe pre-amplification reaction, the products of the respective reactionare amplified. Preferred methods of amplification include the polymerasechain reaction (PCR) or rolling circle amplification (RCA) as is knownin the art. Methods include contacting the reaction products withprimers that hybridize to the priming sites that are included in theinitial target probe. That is, the initial target probe contains primingsites, preferably universal priming sites for the amplification of theproducts. Accordingly, the amplification primers are universal primers;that is the primers to amplify the different detection or genotypingproducts contain the same sequence.

[0164] In a preferred embodiment, the amplification technique is PCR.The polymerase chain reaction (PCR) is widely used and described, andinvolves the use of primer extension combined with thermal cycling toamplify a the product of a genotyping or detection assay, or the productof a pre-amplification reaction; see U.S. Pat. Nos. 4,683,195 and4,683,202, and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton,1995, all of which are incorporated by reference. For clarity, thesereaction products are referred to herein as amplification templates.

[0165] In general, PCR may be briefly described as follows. The doublestranded amplification template is denatured, generally by raising thetemperature, and then cooled in the presence of an excess of a PCRprimer, which then hybridizes to the first priming site, i.e. universalpriming site when universal priming sites are included in the initialtarget probe(s). A DNA polymerase then acts to extend the primer withdNTPs, resulting in the synthesis of a new strand forming ahybridization complex. The sample is then heated again, to disassociatethe hybridization complex, and the process is repeated. By using asecond PCR primer for the complementary target strand that hybridizes tothe second universal priming site, rapid and exponential amplificationoccurs. Thus PCR steps are denaturation, annealing and extension. Theparticulars of PCR are well known, and include the use of a thermostablepolymerase such as Taq I polymerase and thermal cycling. Suitable DNApolymerases include, but are not limited to, the Klenow fragment of DNApolymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNApolymerase and Phi29 DNA polymerase.

[0166] The reaction is initiated by contacting the amplificationtemplates with a solution comprising the primers (universal primers whenapplicable), a polymerase and a set of nucleotides. By “nucleotide” inthis context herein is meant a deoxynucleoside-triphosphate (also calleddeoxynucleotides or dNTPs, e.g. dATP, dTTP, dCTP and dGTP). In someembodiments, as outlined below, one or more of the nucleotides maycomprise a detectable label, which may be either a primary or asecondary label. In addition, the nucleotides may be nucleotide analogs,depending on the configuration of the system. Similarly, the primers maycomprise a primary or secondary label.

[0167] Accordingly, the PCR reaction requires at least one PCR primer, apolymerase, and a set of dNTPs. As outlined herein, the primers maycomprise the label, or one or more of the dNTPs may comprise a label.

[0168] In a preferred embodiment, the methods of the invention include arolling circle amplification (RCA) step. This may be done in severalways. In one embodiment, either single target probes or ligated probescan be used in the detection or genotyping part of the assay asdescribed above, followed by RCA instead of PCR. That is, RCA is used toamplify the product of the detection or genotyping reaction orpre-amplification reaction. Alternatively, and more preferably, the RCAreaction forms part of the genotyping reaction as described above andcan be used for both detection or genotyping and amplification in themethods of the reaction.

[0169] In a preferred embodiment, the methods rely on rolling circleamplification. “Rolling circle amplification” is based on extension of acircular probe that has hybridized to a target sequence when used in thedetection or genotyping reaction, or amplification templates whenamplifying the products of the reaction. A polymerase is added thatextends the probe sequence. As the circular probe has no terminus, thepolymerase repeatedly extends the circular probe resulting inconcatamers of the circular probe. As such, the probe is amplified.Rolling-circle amplification is generally described in Baner et al.(1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad.Sci. USA 88:189-193; and Lizardi et al. (1998) Nat. Genet. 19:225-232,all of which are incorporated by reference in their entirety.

[0170] Labeling of the amplicon can be accomplished in a variety ofways; for example, the polymerase may incorporate labeled nucleotides,or alternatively, a label probe is used that is substantiallycomplementary to a portion of the RCA probe and comprises at least onelabel is used, as is generally outlined herein.

[0171] The polymerase can be any polymerase, but is preferably onelacking 3′ exonuclease activity (3′ exo-). Examples of suitablepolymerase include but are not limited to exonuclease minus DNAPolymerase I large (Klenow) Fragment, Phi29 DNA polymerase, Taq DNAPolymerase and the like. In addition, in some embodiments, a polymerasethat will replicate single-stranded DNA (i.e. without a primer forming adouble stranded section) can be used.

[0172] In a preferred embodiment, the RCA probe contains an adaptersequence as outlined herein, with adapter capture probes on the array,for example on a microsphere when microsphere arrays are being used.Alternatively, unique portions of the RCA probes, for example all orpart of the sequence corresponding to the target sequence, can be usedto bind to a capture probe.

[0173] In a preferred embodiment, the padlock probe contains arestriction site. The restriction endonuclease site allows for cleavageof the long concatamers that are typically the result of RCA intosmaller individual units that hybridize either more efficiently orfaster to surface bound capture probes. Thus, following RCA, the productnucleic acid is contacted with the appropriate restriction endonuclease.This results in cleavage of the product nucleic acid into smallerfragments. The fragments are then hybridized with the capture probe thatis immobilized resulting in a concentration of product fragments ontothe microsphere. Again, as outlined herein, these fragments can bedetected in one of two ways: either labelled nucleotides areincorporated during the replication step, or an additional label probeis added.

[0174] Thus, in a preferred embodiment, the padlock probe comprises alabel sequence; i.e. a sequence that can be used to bind label probesand is substantially complementary to a label probe. In one embodiment,it is possible to use the same label sequence and label probe for allpadlock probes on an array; alternatively, each padlock probe can have adifferent label sequence.

[0175] The padlock probe also contains a priming site for priming theRCA reaction. That is, each padlock probe comprises a sequence to whicha primer nucleic acid hybridizes forming a template for the polymerase.The primer can be found in any portion of the circular probe. In apreferred embodiment, the primer is located at a discrete site in theprobe. In this embodiment, the primer site in each distinct padlockprobe is identical, e.g. is a universal priming site, although this isnot required. Advantages of using primer sites with identical sequencesinclude the ability to use only a single primer oligonucleotide to primethe RCA assay with a plurality of different hybridization complexes.That is, the padlock probe hybridizes uniquely to the target nucleicacid to which it is designed. A single primer hybridizes to all of theunique hybridization complexes forming a priming site for thepolymerase. RCA then proceeds from an identical locus within each uniquepadlock probe of the hybridization complexes.

[0176] In an alternative embodiment, the primer site can overlap,encompass, or reside within any of the above-described elements of thepadlock probe. That is, the primer can be found, for example,overlapping or within the restriction site or the identifier sequence.In this embodiment, it is necessary that the primer nucleic acid isdesigned to base pair with the chosen primer site.

[0177] In a preferred embodiment the RCA is performed in solutionfollowed by restriction endonuclease cleavage of the RCA product. Thecleaved product is then applied to an array comprising beads, each beadcomprising a probe complementary to the adapter sequence located in thepadlock probe or complementary to the target specific sequence. Theamplified adapter sequence correlates with a particular target nucleicacid. Thus the incorporation of an endonuclease site allows thegeneration of short, easily hybridizable sequences. Furthermore, theunique adapter sequence in each rolling circle padlock probe sequenceallows diverse sets of nucleic acid sequences to be analyzed in parallelon an array, since each sequence is resolved on the basis ofhybridization specificity.

[0178] Thus, the present invention provides for the generation ofamplicons.

[0179] In a preferred embodiment, the amplicons are labeled with adetection label. By “detection label” or “detectable label” herein ismeant a moiety that allows detection. This may be a primary label or asecondary label. Accordingly, detection labels may be primary labels(i.e. directly detectable) or secondary labels (indirectly detectable).

[0180] In a preferred embodiment, the detection label is a primarylabel. A primary label is one that can be directly detected, such as afluorophore. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal labels; and c) colored or luminescent dyes. Labelscan also include enzymes (horseradish peroxidase, etc.) and magneticparticles. Preferred labels include chromophores or phosphors but arepreferably fluorescent dyes. Suitable dyes for use in the inventioninclude, but are not limited to, fluorescent lanthanide complexes,including those of Europium and Terbium, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,quantum dots (also referred to as “nanocrystals”: see U.S. Ser. No.09/315,584, hereby incorporated by reference), pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cy dyes (Cy3, Cy5,etc.), alexa dyes, phycoerythin, bodipy, and others described in the 6thEdition of the Molecular Probes Handbook by Richard P. Haugland, herebyexpressly incorporated by reference.

[0181] In a preferred embodiment, a secondary detectable label is used.A secondary label is one that is indirectly detected; for example, asecondary label can bind or react with a primary label for detection,can act on an additional product to generate a primary label (e.g.enzymes), or may allow the separation of the compound comprising thesecondary label from unlabeled materials, etc. Secondary labels include,but are not limited to, one of a binding partner pair such asbiotin/streptavidin; chemically modifiable moieties; nucleaseinhibitors, enzymes such as horseradish peroxidase, alkalinephosphatases, lucifierases, etc.

[0182] In a preferred embodiment, the secondary label is a bindingpartner pair. For example, the label may be a hapten or antigen, whichwill bind its binding partner. In a preferred embodiment, the bindingpartner can be attached to a solid support to allow separation ofextended and non-extended primers. For example, suitable binding partnerpairs include, but are not limited to: antigens (such as proteins(including peptides)) and antibodies (including fragments thereof (FAbs,etc.)); proteins and small molecules, including biotin/streptavidin;enzymes and substrates or inhibitors; other protein-protein interactingpairs; receptor-ligands; and carbohydrates and their binding partners.Nucleic acid nucleic acid binding proteins pairs are also useful. Ingeneral, the smaller of the pair is attached to the NTP forincorporation into the primer. Preferred binding partner pairs include,but are not limited to, biotin (or imino-biotin) and streptavidin,digeoxinin and Abs, and Prolinx™ reagents (seewww.prolinxinc.com/ie4/home.hmtl).

[0183] In a preferred embodiment, the binding partner pair comprisesbiotin or imino-biotin and streptavidin. Imino-biotin is particularlypreferred as imino-biotin disassociates from streptavidin in pH 4.0buffer while biotin requires harsh denaturants (e.g. 6 M guanidiniumHCl, pH 1.5 or 90% formamide at 95° C.).

[0184] In a preferred embodiment, the binding partner pair comprises aprimary detection label (for example, attached to the NTP and thereforeto the amplicon) and an antibody that will specifically bind to theprimary detection label. By “specifically bind” herein is meant that thepartners bind with specificity sufficient to differentiate between thepair and other components or contaminants of the system. The bindingshould be sufficient to remain bound under the conditions of the assay,including wash steps to remove non-specific binding. In someembodiments, the dissociation constants of the pair will be less thanabout 10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ beingpreferred and less than about 10⁻⁷-10⁻⁹ M⁻¹ being particularlypreferred.

[0185] In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the nucleic acid. The functionalgroup can then be subsequently labeled with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups, with amino groupsand thiol groups being particularly preferred. For example, primarylabels containing amino groups can be attached to secondary labelscomprising amino groups, for example using linkers as are known in theart; for example, homo-or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).

[0186] As outlined herein, labeling can occur in a variety of ways, aswill be appreciated by those in the art. In general, labeling can occurin one of three ways: labels are incorporated into primers such that theamplification reaction results in amplicons that comprise the labels;labels are attached to dNTPs and incorporated by the polymerase into theamplicons; or the amplicons comprise a label sequence that is used tohybridize a label probe, and the label probe comprises the labels. Itshould be noted that in the latter case, the label probe can be addedeither before the amplicons are contacted with an array or afterwards.

[0187] A preferred embodiment utilizes one primer comprising a biotin,that is used to bind a fluorescently labeled streptavidin.

[0188] Re-Use of Target Nucleic Acids

[0189] Upon completion of a first round of analyses, i.e. detection orgenotyping, the modified probes are removed from the target nucleicacids as described above. Once removed, the immobilized target nucleicacids are contacted with a second set of target probes for subsequentanalyses. That is, the invention provides methods of reusing immobilizedtarget nucleic acids.

[0190] Re-using immobilized target nucleic acids has a number ofadvantages. First, the method maximizes the information obtained from asingle sample or starting material. Accordingly, the invention providesa method of increasing the information obtained from an immobilizedtarget nucleic acid. As described above, the method includes performinga first analysis with a first probe or probe set, removing the modifiedprobe(s) and performing a second analysis with a second probe or probeset. While there is no theoretical limit to the number of reactions orreiterative uses performed on an immobilized target, in one embodimentthe target nucleic acid is re-used at least twice, more preferably atleast 10 times, and most preferably at least 20 times.

[0191] In a preferred embodiment, the signal obtained from subsequentanalyses is not diminished when compared to the first reaction, althoughin some instances the signal is reduced. Preferably the signal is notreduced more than 40%, with not more than 20% more preferred and notmore than 10% most preferred. That is, when comparing a signal from asecond analysis with the signal from the first analysis, the signal fromthe second preferably is not less than 60% of that obtained from thefirst, more preferably not less than 80% less than the signal from thefirst and most preferably not less than 90% of that obtained from thefirst signal. In a preferred embodiment, the signal from each successiveanalysis is not diminished relative to the signal obtained from theprevious assay.

[0192] Detection of Amplicons

[0193] Once removed from the target sequence and after the optionalamplification and/or pre-amplification procedure described above, theprobe or probes from either the first or any of the subsequent assays asdescribed above or the amplicons are analyzed to detect the productand/or identify the nucleotide at the detection position of the targetsequence. That is, the above described genotyping assays oramplification assays results in the production of modified target probesas an indication of the nucleotide at the detection position of targetsequence. When the modified target probes are amplified as describedabove, the products are amplicons. Detection is accomplished in avariety of ways, but is preferably accomplished by immobilization of theamplicons, or modified target probes to a solid support.

[0194] Alternatively, detection is accomplished by non-array basedmethods such as mass spectrometry of capillary electrophoresis. Inaddition, the amplicons can be detected by liquid array, i.e.three-dimensional array methods such as flow cytometry.

[0195] When mass spectrometry is the detection method, the amplicons areapplied to a mass spectrometer where their unique masses are detected asis known in the art.

[0196] When capillary electrophoresis is the detection method, ampliconsare applied to a capillary electrophoresis device, and the amplicons arecharacterized by their electrophoretic mobility. The DNA in thecapillary electrophoresis device could be detected electrically at oneor more locations along the electrophoresis channel. Preferably,however, the DNA is detected optically.

[0197] When flow cytometry is the detection method, amplicons areimmobilized to a support such as a microsphere as described herein. Themicrospheres are applied to a flow cytometer and the amplicons aredetected optically as described herein.

[0198] Accordingly, upon completion of the detection or amplificationreactions, target probes or readout probes as described herein becomemodified target probes or readout probes. Thus, modified target orreadout probes can be either directly detected or subject topre-amplification and/or amplification as described herein. The productof the pre-amplification reaction is a pre-amplification product. Theproduct of the amplification reaction is an amplicon. While thediscussion below is applicable to detection of both reaction products,i.e. modified target probes or amplicons, the terminology will bedirected to detection of amplicons. Accordingly, for detection ofamplicons, the present invention provides array compositions comprisingarray substrates with surfaces comprising discrete sites. The presentinvention provides methods and compositions useful in the detection ofnucleic acids, particularly the labeled amplicons outlined herein. As ismore fully outlined below, preferred systems of the invention work asfollows. Amplicons are attached (via hybridization) to an array site.This attachment can be either directly to a capture probe on the arraysurface, through the use of adapters, or indirectly, using captureextender probes as outlined herein. In some embodiments, the ampliconitself comprises the labels. Alternatively, a label probe is then added,forming a detection complex. By “detection complex” is meant an ampliconhybridized with a label probe for detection. The attachment of the labelprobe may be direct (i.e. hybridization to a portion of the amplicon),or indirect (i.e. hybridization to an amplifier probe that hybridizes tothe amplicon), with all the required nucleic acids forming an assaycomplex.

[0199] Accordingly, the present invention also contemplates the use ofarray compositions comprising at least a first array substrate with asurface comprising individual sites. By “array” or “biochip” herein ismeant a plurality of nucleic acids in an array format; the size of thearray will depend on the composition and end use of the array. Nucleicacids arrays are known in the art, and can be classified in a number ofways; both ordered arrays (e.g. the ability to resolve chemistries atdiscrete sites), and random arrays are included. Ordered arrays include,but are not limited to, those made using photolithography techniques(Affymetrix GeneChip™), spotting techniques (Synteni and others),printing techniques (Hewlett Packard and Rosetta), three dimensional“gel pad” arrays, etc. A preferred embodiment utilizes microspheres on avariety of array substrates including fiber optic bundles, as areoutlined in PCTs US98/21193, PCT US99/14387 and PCT US98/05025;WO98/50782; and U.S. Ser. Nos. 09/287,573, 09/151,877, 09/256,943,09/316,154, No. 60/119,323, Ser. No. 09/315,584; all of which areexpressly incorporated by reference. While much of the discussion belowis directed to the use of microsphere arrays on fiber optic bundles, anyarray format of nucleic acids on solid supports may be utilized.

[0200] Arrays containing from about 2 different bioactive agents (e.g.different beads, when beads are used) to many millions can be made, withvery large arrays being possible. Generally, the array will comprisefrom two to as many as a billion or more, depending on the size of thebeads and the array substrate, as well as the end use of the array, thusvery high density, high density, moderate density, low density and verylow density arrays may be made. Preferred ranges for very high densityarrays are from about 10,000,000 to about 2,000,000,000, with from about100,000,000 to about 1,000,000,000 being preferred (all numbers being insquare cm). High density arrays range about 100,000 to about 10,000,000,with from about 1,000,000 to about 5,000,000 being particularlypreferred. Moderate density arrays range from about 10,000 to about100,000 being particularly preferred, and from about 20,000 to about50,000 being especially preferred. Low density arrays are generally lessthan 10,000, with from about 1,000 to about 5,000 being preferred. Verylow density arrays are less than 1,000, with from about 10 to about 1000being preferred, and from about 100 to about 500 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single bioactive agent may be made as well. In addition, insome arrays, multiple array substrates may be used, either of differentor identical compositions. Thus for example, large arrays may comprise aplurality of smaller array substrates.

[0201] In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 μm orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 40,000 or more (in someinstances, 1 million) different elements (e.g. fibers and beads) in a 1mm² fiber optic bundle, with densities of greater than 25,000,000individual beads and fibers (again, in some instances as many as 50-100million) per 0.5 cm² obtainable (4 million per square cm for 5μcenter-to-center and 100 million per square cm for 1μ center-to-center).

[0202] By “array substrate” or “array solid support” or othergrammatical equivalents herein is meant any material that can bemodified to contain discrete individual sites appropriate for theattachment or association of beads and is amenable to at least onedetection method. As will be appreciated by those in the art, the numberof possible array substrates is very large. Possible array substratesinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon, etc.), polysaccharides, nylon or nitrocellulose,resins, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, plastics, optical fiberbundles, and a variety of other polymers. In general, the arraysubstrates allow optical detection and do not themselves appreciablyfluoresce.

[0203] Generally the array substrate is flat (planar), although as willbe appreciated by those in the art, other configurations of arraysubstrates may be used as well; for example, three dimensionalconfigurations can be used, for example by embedding the beads in aporous block of plastic that allows sample access to the beads and usinga confocal microscope for detection. Similarly, the beads may be placedon the inside surface of a tube, for flow-through sample analysis tominimize sample volume. Preferred array substrates include optical fiberbundles as discussed below, and flat planar array substrates such aspaper, glass, polystyrene and other plastics and acrylics.

[0204] In a preferred embodiment, the array substrate is an opticalfiber bundle or array, as is generally described in U.S. Ser. Nos.08/944,850 and 08/519,062, PCT US98/05025, and PCT US98/09163, all ofwhich are expressly incorporated herein by reference. Preferredembodiments utilize preformed unitary fiber optic arrays. By “preformedunitary fiber optic array” herein is meant an array of discreteindividual fiber optic strands that are co-axially disposed and joinedalong their lengths. The fiber strands are generally individually clad.However, one thing that distinguished a preformed unitary array fromother fiber optic formats is that the fibers are not individuallyphysically manipulatable; that is, one strand generally cannot bephysically separated at any point along its length from another fiberstrand.

[0205] Generally, the arrayed array compositions of the invention can beconfigured in several ways; see for example U.S. Ser. No. 09/473,904,hereby expressly incorporated by reference. In a preferred embodiment,as is more fully outlined below, a “one component” system is used. Thatis, a first array substrate comprising a plurality of assay locations(sometimes also referred to herein as “assay wells”), such as amicrotiter plate, is configured such that each assay location containsan individual array. That is, the assay location and the array locationare the same. For example, the plastic material of the microtiter platecan be formed to contain a plurality of “bead wells” in the bottom ofeach of the assay wells. Beads containing the capture probes of theinvention can then be loaded into the bead wells in each assay locationas is more fully described below.

[0206] Alternatively, a “two component” system can be used. In thisembodiment, the individual arrays are formed on a second arraysubstrate, which then can be fitted or “dipped” into the firstmicrotiter plate substrate. A preferred embodiment utilizes fiber opticbundles as the individual arrays, generally with “bead wells” etchedinto one surface of each individual fiber, such that the beadscontaining the capture probes are loaded onto the end of the fiber opticbundle. The composite array thus comprises a number of individual arraysthat are configured to fit within the wells of a microtiter plate.

[0207] By “composite array” or “combination array” or grammaticalequivalents herein is meant a plurality of individual arrays, asoutlined above. Generally the number of individual arrays is set by thesize of the microtiter plate used; thus, 96 well, 384 well and 1536 wellmicrotiter plates utilize composite arrays comprising 96, 384 and 1536individual arrays, although as will be appreciated by those in the art,not each microtiter well need contain an individual array. It should benoted that the composite arrays can comprise individual arrays that areidentical, similar or different. That is, in some embodiments, it may bedesirable to do the same 2,000 assays on 96 different samples;alternatively, doing 192,000 experiments on the same sample (i.e. thesame sample in each of the 96 wells) may be desirable. Alternatively,each row or column of the composite array could be the same, forredundancy/quality control. As will be appreciated by those in the art,there are a variety of ways to configure the system. In addition, therandom nature of the arrays may mean that the same population of beadsmay be added to two different array surfaces, resulting in substantiallysimilar but perhaps not identical arrays.

[0208] At least one surface of the array substrate is modified tocontain discrete, individual sites for later association ofmicrospheres. These sites may comprise physically altered sites, i.e.physical configurations such as wells or small depressions in the arraysubstrate that can retain the beads, such that a microsphere can rest inthe well, or the use of other forces (magnetic or compressive) orchemically altered or active sites, such as chemically functionalizedsites, electrostatically altered sites, hydrophobically/hydrophilicallyfunctionalized sites, spots of adhesive, etc.

[0209] The sites may be a pattern, i.e. a regular design orconfiguration, or randomly distributed. A preferred embodiment utilizesa regular pattern of sites such that the sites may be addressed in theX-Y coordinate plane. “Pattern” in this sense includes a repeating unitcell, preferably one that allows a high density of beads on the arraysubstrate. However, it should be noted that these sites may not bediscrete sites. That is, it is possible to use a uniform surface ofadhesive or chemical functionalities, for example, that allows theattachment of beads at any position. That is, the surface of the arraysubstrate is modified to allow attachment of the microspheres atindividual sites, whether or not those sites are contiguous ornon-contiguous with other sites. Thus, the surface of the arraysubstrate may be modified such that discrete sites are formed that canonly have a single associated bead, or alternatively, the surface of thearray substrate is modified and beads may go down anywhere, but they endup at discrete sites. That is, while beads need not occupy each site onthe array, no more than one bead occupies each site.

[0210] In a preferred embodiment, the surface of the array substrate ismodified to contain wells, i.e. depressions in the surface of the arraysubstrate. This may be done as is generally known in the art using avariety of techniques, including, but not limited to, photolithography,stamping techniques, molding techniques and microetching techniques. Aswill be appreciated by those in the art, the technique used will dependon the composition and shape of the array substrate.

[0211] In a preferred embodiment, physical alterations are made in asurface of the array substrate to produce the sites. In a preferredembodiment, the array substrate is a fiber optic bundle and the surfaceof the array substrate is a terminal end of the fiber bundle, as isgenerally described in Ser. Nos. 08/818,199 and 09/151,877, both ofwhich are hereby expressly incorporated by reference. In thisembodiment, wells are made in a terminal or distal end of a fiber opticbundle comprising individual fibers. In this embodiment, the cores ofthe individual fibers are etched, with respect to the cladding, suchthat small wells or depressions are formed at one end of the fibers. Therequired depth of the wells will depend on the size of the beads to beadded to the wells.

[0212] Generally in this embodiment, the microspheres are non-covalentlyassociated in the wells, although the wells may additionally bechemically functionalized as is generally described below, cross-linkingagents may be used, or a physical barrier may be used, i.e. a film ormembrane over the beads.

[0213] In a preferred embodiment, the surface of the array substrate ismodified to contain chemically modified sites, that can be used toattach, either covalently or non-covalently, the microspheres of theinvention to the discrete sites or locations on the array substrate.“Chemically modified sites” in this context includes, but is not limitedto, the addition of a pattern of chemical functional groups includingamino groups, carboxy groups, oxo groups and thiol groups, that can beused to covalently attach microspheres, which generally also containcorresponding reactive functional groups; the addition of a pattern ofadhesive that can be used to bind the microspheres (either by priorchemical functionalization for the addition of the adhesive or directaddition of the adhesive); the addition of a pattern of charged groups(similar to the chemical functionalities) for the electrostaticattachment of the microspheres, i.e. when the microspheres comprisecharged groups opposite to the sites; the addition of a pattern ofchemical functional groups that renders the sites differentiallyhydrophobic or hydrophilic, such that the addition of similarlyhydrophobic or hydrophilic microspheres under suitable experimentalconditions will result in association of the microspheres to the siteson the basis of hydroaffinity. For example, the use of hydrophobic siteswith hydrophobic beads, in an aqueous system, drives the association ofthe beads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

[0214] In some embodiments, the beads are not associated with an arraysubstrate. That is, the beads are in solution or are not distributed ona patterned substrate.

[0215] In a preferred embodiment, the compositions of the inventionfurther comprise a population of microspheres. By “population” herein ismeant a plurality of beads as outlined above for arrays. Within thepopulation are separate subpopulations, which can be a singlemicrosphere or multiple identical microspheres. That is, in someembodiments, as is more fully outlined below, the array may contain onlya single bead for each capture probe; preferred embodiments utilize aplurality of beads of each type.

[0216] By “microspheres” or “beads” or “particles” or grammaticalequivalents herein is meant small discrete particles. The composition ofthe beads will vary, depending on the class of capture probe and themethod of synthesis. Suitable bead compositions include those used inpeptide, nucleic acid and organic moiety synthesis, including, but notlimited to, plastics, ceramics, glass, polystyrene, methylstyrene,acrylic polymers, paramagnetic materials, thoria sol, carbon graphite,titanium dioxide, latex or cross-linked dextrans such as Sepharose,cellulose, nylon, cross-linked micelles and Teflon may all be used.“Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind. is ahelpful guide.

[0217] The beads need not be spherical; irregular particles may be used.In addition, the beads may be porous, thus increasing the surface areaof the bead available for either capture probe attachment or tagattachment. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200microns being preferred, and from about 0.5 to about 5 micron beingparticularly preferred, although in some embodiments smaller beads maybe used.

[0218] It should be noted that a key component of the invention is theuse of an array substrate/bead pairing that allows the association orattachment of the beads at discrete sites on the surface of the arraysubstrate, such that the beads do not move during the course of theassay.

[0219] Each microsphere comprises a capture probe, although as will beappreciated by those in the art, there may be some microspheres which donot contain a capture probe, depending on the synthetic methods.

[0220] Attachment of the nucleic acids may be done in a variety of ways,as will be appreciated by those in the art, including, but not limitedto, chemical or affinity capture (for example, including theincorporation of derivatized nucleotides such as AminoLink orbiotinylated nucleotides that can then be used to attach the nucleicacid to a surface, as well as affinity capture by hybridization),cross-linking, and electrostatic attachment, etc. In a preferredembodiment, affinity capture is used to attach the nucleic acids to thebeads. For example, nucleic acids can be derivatized, for example withone member of a binding pair, and the beads derivatized with the othermember of a binding pair. Suitable binding pairs are as described hereinfor IBL/DBL pairs. For example, the nucleic acids may be biotinylated(for example using enzymatic incorporate of biotinylated nucleotides,for by photoactivated cross-linking of biotin). Biotinylated nucleicacids can then be captured on streptavidincoated beads, as is known inthe art. Similarly, other hapten-receptor combinations can be used, suchas digoxigenin and anti-digoxigenin antibodies. Alternatively, chemicalgroups can be added in the form of derivatized nucleotides, that canthem be used to add the nucleic acid to the surface.

[0221] Preferred attachments are covalent, although even relatively weakinteractions (i.e. non-covalent) can be sufficient to attach a nucleicacid to a surface, if there are multiple sites of attachment per eachnucleic acid. Thus, for example, electrostatic interactions can be usedfor attachment, for example by having beads carrying the opposite chargeto the bioactive agent.

[0222] Similarly, affinity capture utilizing hybridization can be usedto attach nucleic acids to beads.

[0223] Alternatively, chemical crosslinking may be done, for example byphotoactivated crosslinking of thymidine to reactive groups, as is knownin the art.

[0224] In a preferred embodiment, each bead comprises a single type ofcapture probe, although a plurality of individual capture probes arepreferably attached to each bead. Similarly, preferred embodimentsutilize more than one microsphere containing a unique capture probe;that is, there is redundancy built into the system by the use ofsubpopulations of microspheres, each microsphere in the subpopulationcontaining the same capture probe.

[0225] As will be appreciated by those in the art, the capture probesmay either be synthesized directly on the beads, or they may be made andthen attached after synthesis. In a preferred embodiment, linkers areused to attach the capture probes to the beads, to allow both goodattachment, sufficient flexibility to allow good interaction with thetarget molecule, and to avoid undesirable binding reactions.

[0226] In a preferred embodiment, the capture probes are synthesizeddirectly on the beads. As is known in the art, many classes of chemicalcompounds are currently synthesized on solid supports, such as peptides,organic moieties, and nucleic acids. It is a relatively straightforwardmatter to adjust the current synthetic techniques to use beads.

[0227] In a preferred embodiment, the capture probes are synthesizedfirst, and then covalently attached to the beads. As will be appreciatedby those in the art, this will be done depending on the composition ofthe capture probes and the beads. The functionalization of solid supportsurfaces such as certain polymers with chemically reactive groups suchas thiols, amines, carboxyls, etc. is generally known in the art.Accordingly, “blank” microspheres may be used that have surfacechemistries that facilitate the attachment of the desired functionalityby the user. Some examples of these surface chemistries for blankmicrospheres include, but are not limited to, amino groups includingaliphatic and aromatic amines, carboxylic acids, aldehydes, amides,chloromethyl groups, hydrazide, hydroxyl groups, sulfonates andsulfates.

[0228] In general, the methods of making the arrays and of decoding thearrays is done to maximize the number of different candidate agents thatcan be uniquely encoded. The compositions of the invention may be madein a variety of ways. In general, the arrays are made by adding asolution or slurry comprising the beads to a surface containing thesites for attachment of the beads. This may be done in a variety ofbuffers, including aqueous and organic solvents, and mixtures. Thesolvent can evaporate, and excess beads are removed.

[0229] In a preferred embodiment, when non-covalent methods are used toassociate the beads with the array, a novel method of loading the beadsonto the array is used. This method comprises exposing the array to asolution of particles (including microspheres and cells) and thenapplying energy, e.g. agitating or vibrating the mixture. In a preferredembodiment when the array substrate is a fiber optic bundle, the arraysubstrate is tapped into the beads. That is, the energy is tapping. Thisresults in an array comprising more tightly associated particles, as theagitation is done with sufficient energy to cause weakly-associatedbeads to fall off (or out, in the case of wells). These sites are thenavailable to bind a different bead. In this way, beads that exhibit ahigh affinity for the sites are selected. Arrays made in this way havetwo main advantages as compared to a more static loading: first of all,a higher percentage of the sites can be filled easily, and secondly, thearrays thus loaded show a substantial decrease in bead loss duringassays. Thus, in a preferred embodiment, these methods are used togenerate arrays that have at least about 50% of the sites filled, withat least about 75% being preferred, and at least about 90% beingparticularly preferred. Similarly, arrays generated in this mannerpreferably lose less than about 20% of the beads during an assay, withless than about 10% being preferred and less than about 5% beingparticularly preferred.

EXAMPLES Example 1 Solid Phase Genomic DNA is Reusable

[0230] Protocol: Begin with 1 ug gDNA on 50 ug of beads. (Homozygous1958 and 2180).

[0231] 1. Hybridize OLA oligos for locus 1958. Ligate. Elute productswith NaOH. Amplify by PCR.

[0232] 2. Hybridize OLA oligos for locus 2180. Ligate. Elute productswith NaOH. Amplify by PCR.

[0233] 3. Repeat steps 1 and 2 through 6 cycles.

[0234] Analyze by agarose gel electrophoresis. See FIG. 5.

[0235] Conclusion: Genomic DNA immobilized on a solid phase is reusableat least six times. Ligase allele selectivity is quite good.

Example 2 Genomic DNA on Magnetic Particles is Reusable in theOligonucleotide Ligation Assay

[0236] Protocol: To beaded DNA-hybridize 48 SNP oligos, wash, ligate,elute, PCR amplify.

[0237] Store overnight. Mix controls. Hybridize 48 SNP oligos, wash,ligate, elute, PCR amplify.

[0238] Analyze by agarose gel electrophoresis. See FIGS. 6A and B.

We claim:
 1. A method comprising: a. providing a composition comprisingfirst primers and target nucleic acid, wherein either said first primersro said target nucleic acid is immobilized to at least one solidsupport; b. performing a first analysis of said target nucleic acid,said first analysis comprising: i) contacting said first primers withsaid target nucleic acid whereby at least one of said first primershybridizes with said target nucleic acid; ii) removing unhybridizedfirst primers; and iii) contacting said hybridized first primers with anenzyme such that said hybridized first primers are modified formingfirst modified primers, whereby said target nucleic acid is notconsumed; and c. performing a second analysis of said target nucleicacid.
 2. The method according to claim 1 wherein said second analysiscomprises: a. contacting second primers with said target nucleic acidwhereby at least one of said second primers hybridizes with said targetnucleic acid; b. removing unhybridized second primers; and c. contactingsaid hybridized second primers with an enzyme such that said hybridizedsecond primers are modified forming second modified primers.
 3. Themethod according to claim 2, further comprising detecting said first andsecond modified primers.
 4. The method according to claim 2, furthercomprising amplifying said first and second modified primers to formfirst and second amplicons.
 5. The method according to claim 4, furthercomprising detecting said first and second amplicons.
 6. The methodaccording to claim 5, wherein said first and second amplicons compriselabels.
 7. The method according to claim 6, wherein said first andsecond amplicons are labeled during said amplification.
 8. The methodaccording to claim 1, wherein said target nucleic acid comprises genomicDNA.
 9. The method according to claim 8, wherein said genomic DNAcomprises at least one copy of the genomic DNA from an organism.
 10. Themethod according to claim 9, wherein said organism is selected fromhumans, mice, pigs, cows, bacteria, viruses or plants.
 11. The methodaccording to claim 1, wherein at least one of said first and secondprimers comprises an adapter sequence.
 12. A method comprising: a.providing a composition comprising first primers and target nucleic acidwherein said first primers are ligation primers; b. hybridizing saidfirst ligation primers with said target nucleic acid to form firstligation complexes, whereby said first ligation primers hybridize tosaid target nucleic acid flanking a first target sequence; c. removingunhybridized ligation primers; d. contacting said first ligationcomplexes with a ligation enzyme, whereby when said first ligationprimers are complementary to said first target sequences, said ligationenzyme ligates said first ligation primers generating first ligationproducts; e. removing said first ligation products from said targetnucleic acid; f. hybridizing said target nucleic acid with secondligation primers to form second ligation complexes, whereby said secondligation primers hybridize to said target nucleic acid flanking a secondtarget sequence; g. contacting said second ligation complex with aligation enzyme, whereby when said second ligation primers arecomplementary to said second target sequence, said ligation enzymeligates said second ligation primers generating second ligationproducts.
 13. The method according to claim 12 further comprising: h.contacting said first and second ligation products with amplificationprimers, nucleotides and amplification enzyme to form first and secondamplicons; and i. detecting said first and second amplicons.
 14. Themethod according to claim 13, wherein said amplification enzyme is a DNApolymerase and said nucleotides are dNTPs.
 15. The method according toclaim 13, wherein said amplification enzyme is an RNA polymerase andsaid nucleotides are NTPs.
 16. A method of reusing target nucleic acidcomprising: a. providing a composition comprising first primers andtarget nucleic acid, wherein either said first primers or said targetnucleic acid are immobilized on at least one solid support; b.performing a first analysis of said target nucleic acid, said firstanalysis comprising: said first analysis comprising: i) contacting saidfirst primers with said target nucleic acid whereby at least one of saidfirst primers hybridizes with said target nucleic acid; ii) removingunhybridized first primers; and iii) contacting said hybridized firstprimers with an enzyme such that said hybridized first primers aremodified forming first modified primers, whereby said target nucleicacid is not consumed whereby said target nucleic acid is not consumed;and c. reusing said target nucleic acid in a second analysis.
 17. Themethod according to claim 16, wherein said target nucleic acid is reusedat least five times.
 18. The method according to claims 12 or 16,wherein said target nucleic acid is genomic DNA.
 19. The methodaccording to claim 1, 12 or 16, wherein said target nucleic acid isimmobilized on at least one solid support.
 20. The method according toclaim 1, 12 or 16, wherein said first primers are immobilized on atleast one solid support.
 21. the method according to claim 1, 12 or 16,wherein at least 10 different target nucleic acids are analyzed in asingle reaction.
 22. The method according to claim 1, 12 or 16, whereinat least 50 different target nucleic acids are analyzed in a singlereaction.
 23. The method according to claim 1, 12 or 16, wherein atleast 100 different target nucleic acids are analyzed in a singlereaction.